Ultrathin Multilayered Films for Controlled Release of Anionic Reagents

ABSTRACT

Multilayered films, particularly ultrathin multilayered films comprising cationic polymers which are useful for controlled release of anionic species, particularly for controlled release of nucleic acids. The multilayer films herein are useful for temporal controlled released of anionic species, particularly one or more anionic peptides, proteins, nucleic acids or other anionic biological agents. In one aspect, the invention relates to multilayer films which release anionic species (anions) with separate and/or distinct release profiles, particularly wherein the anions are one or more anionic peptides, proteins or nucleic acids or other anionic biological agents

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 60/978,633 filed on Oct. 9, 2007,which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support underGrants EB002746 and EB006820 awarded by the National Institutes ofHealth. The U.S. government has certain rights in the invention

BACKGROUND OF THE INVENTION

Materials that provide control over the release of multiple chemical orbiological agents are of interest in a broad range of biomedical andbiotechnological applications. [J. T. Santini, M. J. Cima, R. Langer,Nature 1999, 397, 335; L. D. Shea, E. Smiley, J. Bonadio, D. J. Mooney,Nat Biotechnol 1999, 17, 551; T. P. Richardson, M. C. Peters, A. B.Ennett, D. J. Mooney, Nat Biotechnol 2001, 19, 1029; W. M. Saltzman, W.L. Olbricht, Nat Rev Drug Discov 2002, 1, 177; A. C. R. Grayson, I. S.Choi, B. M. Tyler, P. P. Wang, H. Brem, M. J. Cima, R. Langer, Nat Mater2003, 2, 767; J. M. Saul, M. P. Linnes, B. D. Ratner, C. M. Giachelli,S. H. Pun, Biomaterials 2007, 28, 4705.] Temporal control over therelease of multiple biological cues, for example, will likely provecritical in applications such as tissue engineering, for which precisecontrol over the administration of multiple different growth factors andother signals is thought to be required to promote the development offunctional tissues. [Shea et al. 1999; Richardson et al. 2001; Saltzmanet al. 2002; Saul et al. 2007.] Such sophisticated levels of control canalso contribute to the development of new tools for basic biomedicalresearch and more effective gene- and protein-based therapies. There issignificant interest in the controlled release of anionic species,particularly anionic polypeptides and nucleic acids, including variousforms of RNA and DNA.

Several recent reports have demonstrated approaches to the encapsulationof proteins or DNA in bulk matrices of degradable polymers or thefabrication of devices that provide control over the release of multipleagents [L. D. Shea, E. Smiley, J. Bonadio, D. J. Mooney, Nat Biotechnol1999, 17, 551; T. P. Richardson, M. C. Peters, A. B. Ennett, D. J.Mooney, Nat Biotechnol 2001, 19, 1029; J. M. Saul, M. P. Linnes, B. D.Ratner, C. M. Giachelli, S. H. Pun, Biomaterials 2007, 28, 4705; Santiniet al. 1999; Satlzman et al. 2002; Grayson et al. 2003.] Despite theseadvances, however, it has proven difficult to design thin films andcoatings that provide control over the release of multiple proteins orDNA constructs with separate and distinct release profiles (e.g., rapidrelease of a first DNA construct, followed by the slower, sustainedrelease of a second DNA construct). This invention relates generally toapproaches to the fabrication of ultrathin polymer-based coatings thatcan be exploited to provide temporal control of release of anionicspecies. At least in part, the invention, relates to approaches to thecontrolled release of two or more anionic species with separate,distinct or both separate and distinct release profiles.

The present work relates to the use of methods developed for thelayer-by-layer assembly of multilayered polyelectrolyte films (or‘polyelectrolyte multilayers’). These methods are entirely aqueous andpermit nanometer-scale control over the structures of thin filmsfabricated from a wide variety of synthetic or natural polyelectrolytes,including DNA. [G. Decher, Science 1997, 277, 1232; P. Bertrand, A.Jonas, A. Laschewsky, R. Legras, Macromol Rapid Comm 2000, 21, 319; P.T. Hammond, Adv Mater 2004, 16, 1271; Z. Y. Tang, Y. Wang, P. Podsiadlo,N. A. Kotov, Adv Mater 2006, 18, 3203; Y. Lvov, G. Decher, G.Sukhorukov, Macromolecules 1993, 26, 5396.]

Multilayers have been designed that release DNA and promotesurface-mediated cell transfection by fabricating films using DNA andcationic polymers that are hydrolytically, enzymatically, or reductivelydegradable. Approaches to the fabrication, characterization, andapplication of DNA-containing multilayers have been reviewed recently.[D. M. Lynn, Soft Matter 2006, 2, 269; D. M. Lynn, Adv Mater 2007, 19,4118.]

It has been reported that DNA can be incorporated into polyelectrolytemultilayers using layer-by-layer methods of assembly [Lvov, et al.,1993] and that it is possible to fabricate films that erode and releaseDNA in aqueous environments if the polycationic components of theseassemblies are designed appropriately. [J. Zhang, L. S. Chua, D. M.Lynn, Langmuir 2004, 20, 8015; C. M. Jewell, J. Zhang, N. J. Fredin, D.M. Lynn, J. Control. Release. 2005, 106, 214; K. F. Ren, J. Ji, J. C.Shen, Biomaterials 2006, 27, 1152; K. F. Ren, J. Ji, J. C. Shen,Bioconjugate Chem. 2006, 17, 77; C. M. Jewell, J. Zhang, N. J. Fredin,M. R. Wolff, T. A. Hacker, D. M. Lynn, Biomacromolecules 2006, 7, 2483;Blacklock, H. Handa, D. Soundara Manickam, G. Mao, A. Mukhopadhyay, D.Oupicky, Biomaterials 2007, 28, 117; J. Chen, S. Huang, W. Lin, R. Zhuo,Small 2007, 3, 636.] For example, it was recently reported thatpolyelectrolyte multilayers fabricated from plasmid DNA andhydrolytically degradable poly(beta-amino ester)s erode when incubatedin physiological media [Zhang, et al. 2004 supra; Jewell, et al. 2005,supra; Jewell, et al. 2006, supra and D. M. Lynn, et al. 2006] and thatobjects coated with these assemblies promote surface-mediatedtransfection when placed in contact with mammalian cells. [Jewell et al.2005, supra; Jewell et al. 2006, supra.]

It has also been reported that enzymatically or reductively degradablecationic polymers can be used to fabricate assemblies that release DNAin the presence of enzymes, reducing agents, or cells. [J. Zhang, et al.2004; Jewell, et al. 2005; Zhang, et al. 2007; Ren, et al. Biomaterials2006; K. F. Ren, J. Ji, J. C. Shen, Bioconjugate Chem. 2006, 17, 77;Blacklock, et al. 2007; Chen et al. 2007; N. Jessel, M.Oulad-Abdelghani, F. Meyer, P. Lavalle, Y. Haikel, P. Schaaf, J. C.Voegel, Proc Natl Acad Sci USA 2006, 103, 8618.]. These studies reportapproaches to promoting film erosion that involve the backbonedegradation of cationic polymers and, in general, lead to films thatrelease DNA relatively rapidly (e.g., over several hours to severaldays).

The present invention, in part, relates to an alternative approach tothe disruption of ionic interactions in these assemblies that provides ameans to extend the release of DNA or other anions over much longerperiods (e.g., several months) particularly in ways that are useful inapplications that require long-term exposure of cells or tissues to DNA.Additionally, the invention relates to approaches for the controlledrelease of two or more anions, particularly from a single multilayerfilm which exhibit rapid short-term release of one anion combined withlong-term release of another anion.

U.S. Pat. No. 7,112,361 relates to decomposable films comprising aplurality of polyelectrolyte bilayers. Related published U.S.application 2007/0020469 reports decomposable films comprising aplurality of polyelectrolyte layers wherein a portion of the bilayerscomprise a second entity selected from a biomolecule, a small molecule,a bioactive agent, and any combination thereof.

U.S. published application 20050027064 relates to charge-dynamicpolymers useful for the delivery of anionic compounds including nucleicacids. The dynamic charge state cationic polymers are designed to havecationic charge densities that decrease by removal of removablefunctional groups from the polymers. The application also relates tocomplexes containing the polymers complexed to a polyanion and methodsfor using the interpolyelectrolyte complexes to deliver anioniccompounds. The application describes compositions comprising a dynamiccharge state cationic polymer, having a polymeric backbone formed frommonomeric units, and having one or more removable functional groupsattached to the polymeric backbone. The cationic charge of the dynamiccharge state cationic polymer decreases when one or more of theremovable functional groups is removed from the polymer. Specificdynamic charge state cationic polymers include those in which thepolymer backbone comprises a polyamine, acrylate or methacrylatepolymer, including polyethyleneimine, poly(propylene imine), poly(allylamine), poly(vinyl amine), poly(amidoamine), or a dendrimer that isfunctionalized with terminal amine groups. The application alsodescribes a method for delivering an anionic compound to a target cellby contacting a composition comprising a interpolyelectrolyte complexcomprising a dynamic charge state cationic polymer complexed to one ormore anions with the target cell thereby allowing the target cell touptake the composition. After entry of the interpolyelectrolyte complexinto the target cell, one or more of the removable functional groups isremoved from the dynamic charge state cationic polymer decreasing thecationic charge of the dynamic charge state cationic polymer andpromoting dissociation of the interpolyelectrolyte complex to releaseone or more anions.

U.S. published application 20060251701 relates to delivery of nucleicacids by polyelectrolyte assemblies formed by layer-by-layer depositionof nucleic acid and polycation and particularly to implantable medicaldevices coated with polyelectrolyte assemblies. Such devices facilitatethe local delivery of a nucleic acid contained in the polyelectrolyteassembly into a cell or tissue at an implantation site.

The following references relate to formation of polyelectrolytemultilayers, dynamic charge state (charge shifting) polymers, release ofanionic polyelectrolytes and/or drug release from thin films:

X. Liu, J. Zhang, and D. M. Lynn, “Polyelectrolyte MultilayersFabricated from ‘Charge-Shifting’ Anionic Polymers: A New Approach toControlled Film Disruption and the Release of Cationic Agents fromSurfaces.” Soft Matter 2008, 4, 1688-1695; C. M. Jewell and D. M. Lynn,“Multilayered Polyelectrolyte Assemblies as Platforms for the Deliveryof DNA and Other Nucleic Acid-Based Therapeutics.” Advanced DrugDelivery Reviews 2008, 60, 979-999; N. J. Fredin, J. Zhang, and D. M.Lynn, “Nanometer-Scale Decomposition of Ultrathin MultilayeredPolyelectrolyte Films.” Langmuir 2007, 23, 2273-2276; J. Zhang, S. I.Montanez, C. M. Jewell, and D. M. Lynn, “Multilayered Films Fabricatedfrom Plasmid DNA and a Side-Chain Functionalized Poly(beta-amino ester):Surface-Type Erosion and Sequential Release of Multiple PlasmidConstructs from Surfaces.” Langmuir 2007, 23, 11139-11146; J. Zhang andD. M. Lynn, “Ultrathin Multilayered Films Assembled from‘Charge-Shifting’ Cationic Polymers: Extended, Long-Term Release ofPlasmid DNA from Surfaces.” Advanced Materials 2007, 19, 4218-4223; D.M. Lynn, “Peeling Back the Layers: Controlled Erosion and TriggeredDisassembly of Multilayered Polyelectrolyte Thin Films.” AdvancedMaterials 2007, 19, 4118-4130; J. Zhang, N. J. Fredin, J. F. Janz, B.Sun, and D. M. Lynn, “Structure/Property Relationships in ErodibleMultilayered Films: Influence of Polycation Structure on ErosionProfiles and the Release of Anionic Polyelectrolytes.” Langmuir 2006,22, 239-245; D. M. Lynn, “Layers of Opportunity: Nanostructured PolymerAssemblies for the Delivery of Macromolecular Therapeutics.” Soft Matter2006, 2, 269-273; K. C. Wood, H. F. Chuang, R. D. Batten, D. M. Lynn,and P. T. Hammond, “Controlling Interlayer Diffusion to AchieveSustained, Multi-Agent Delivery from Layer-by-Layer Films.” Proceedingsof the National Academy of Sciences, USA 2006, 103, 10207-10212; J.Zhang, N. J. Fredin, and D. M. Lynn, “Erosion of Multilayered AssembliesFabricated from Degradable Polyamines: Characterization and Evidence inSupport of a Mechanism that Involves Polymer Hydrorysis.” Journal ofPolymer Science—Part A: Polymer Chemistry 2006, 44, 5161-5173; C. M.Jewell, J. Zhang, N. J. Fredin, M. R. Wolff, T. A. Hacker, and D. M.Lynn, “Release of Plasmid DNA from Intravascular Stents Coated withUltrathin Multilayered Polyelectrolyte Films.” Biomacromolecules 2006,7, 2483-2491; J. Zhang and D. M. Lynn, “Multilayered Films Fabricatedfrom Combinations of Degradable Polyamines Tunable Erosion and Releaseof Anionic Polyelectrolytes.” Macromolecules 2006, 39, 8928-8935; C. M.Jewell, J. Zhang, N. J. Fredin, and D. M. Lynn, “MultilayeredPolyelectrolyte Films Promote the Direct and Localized Delivery of DNAto Cells.” Journal of Controlled Release 2005, 106, 214-223; K. Wood, J.Q. Boedicker, D. M. Lynn, and P. T. Hammond, “Tunable Drug Release fromHydrolytically Degradable Layer-by-Layer Thin Films.” Langmuir 2005, 21,1603-1609; N. J. Fredin, J. Zhang, and D. M. Lynn, “Surface Analysis ofErodible Multilayered Polyelectrolyte Films: Nanometer-Scale Structureand Erosion Profiles.” Langmuir 2005, 21, 5803-5811; and X. Liu, J. W.Yang, A. D. Miller, E. A. Nack, and D. M. Lynn, “Charge-ShiftingCationic Polymers that Promote Self-Assembly and Self-Disassembly withDNA.” Macromolecules 2005, 38, 7907-7914.

There is a need in the art for materials and methods that providecontrol over the release of multiple chemical or biological agents,particularly for controlled release of nucleic acids.

SUMMARY OF THE INVENTION

The invention relates to multilayered films, particularly ultrathinmultilayered films comprising cationic polymers which are useful forcontrolled release of anionic species, particularly for controlledrelease of nucleic acids. The multilayer films herein are useful fortemporal controlled released of anionic species, particularly one ormore anionic peptides, proteins, nucleic acids or other anionicbiological agents

In one aspect, the invention relates to multilayer films which releaseanionic species (anions) with separate and/or distinct release profiles,particularly wherein the anions are one or more anionic peptides,proteins or nucleic acids or other anionic biological agents

In another aspect, the invention relates to multilayer films which areuseful for extended, long-term release of anions, particularly one ormore nucleic acids. In another aspect, the invention relates tomultilayer films which are useful for achieving a combination ofshort-term and long-term controlled release of anions, particularly oneor more anionic peptides, proteins or nucleic acids or other anionicbiological agents. In a specific embodiment, the short-term andlong-term release of anions occurs from a single multiple layer films.

Multilayer films of the invention comprise anion/cationic polymerbilayers. The films can be formed using methods that are entirelyaqueous. Multilayer films can degrade in aqueous solution, at least inpart by degradation of ester linkages of the cationic polymers of one ormore bilayers. Degradation of films and release of anions does notrequire the presence of enzymes or reducing agents.

In one aspect, the invention relates to multilayer polyelectrolytefilms, also called polyelectrolyte assemblies, which are formed from atleast two different cationic charge dynamic polymers, also called chargeshifting polymers, selected from polymers having Formula I:

where:n, m, l, k and i are zero or integers where n+m+l+k+i=N, the totalnumber of repeat units in the polymer;A₁₋₃, B₁₋₃, C₁₋₃, D₁₋₃ and E₁₋₃ are linkers which may be the same oreach may be different;Z₁-Z₅ are most generally covalent bonds which may or may not bedegradable bonds;R₁₋₄ and R₁₁₋₁₉, independently, can be hydrogen, or alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups, all ofwhich are optionally substituted, with the exception that R₁₋₄ are notesters; andR₅-R₁₀ are linkers or covalent bonds which may be the same or each maybe different.

In the polyelectrolyte films formed from cationic polymers of formula Iat least one of the polymers contains one or more hydrolysable estergroups. In specific embodiments, at least one of the polymers employedin the film is biodegradable and biocompatible. In specific embodiments,all of the polymers employed in the film are biodegradable andbiocompatible.

A₁₋₂, B₁₋₂, C₁₋₂, D₁₋₂ and E₁₋₂ are linkers which can be the same ordifferent and can be any substituted or unsubstituted, branched orunbranched chain of carbon atoms and heteroatoms with the exception thatnone of these linkers is substituted with ester groups except asspecifically shown in the formula above. Linkers include those that are1 to 30 atoms long, more preferably 1 to 15 atoms long and yet morepreferably 1 to 6 atoms long. The linkers may be substituted withvarious substituents including, but not limited to, alkyl, alkenyl,alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl,alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano,amide, carbamoyl, thioether, alkylthioether, thiol, and ureido groups.As would be appreciated by one of skill in this art, each of thesegroups may in turn be substituted. A₁₋₂, B₁₋₂, C₁₋₂, D₁₋₂ and E₁₋₂ can,for example, be selected from —(CH₂)_(r)—, —(CH₂)_(r)—NR—,—NR—(CH₂)_(r)—, —(CH₂)_(r)—O—, —O—(CH₂)_(r)—, —(CH₂)_(r)—O—(CH₂)_(s)—,—(CH₂)_(r)—S—, —S—(CH₂)_(r)—, —(CH₂)_(r)—S—(CH₂)_(s), where r and s areintegers ranging from 1 to 30, and where r+s ranges from 2 to 30. Morepreferably r and s range from 1-6 and r+s ranges from 2 to 12.

In specific embodiments of formula I, k and i are zero. In specificembodiments n+m+

+k+i=N=5 to 100,000. More specifically, N can range from 20 to 100,000,from 100 to 100,000, from 1,000 to 100,000 or from 10,000 to 100,000. Ina specific embodiment, all of n, m, k and i are zero. In a specificembodiment, m, n and

are all zero. In specific embodiments, (k+i)/N is 0.01 to 0.1, 0.05 to0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or 0.75 to 1. In specificembodiments, (n+m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1. In specific embodiments, (m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1.

In specific embodiments of formula I, 10% or more of the groups bondedto the amine of the amine side chains of the polymer are ester groups.In specific embodiments, 25% or more of the groups bonded to the amineof the amine side chains of the polymer are ester groups. In specificembodiments, 50% or more of the groups bonded to the amine of the amineside chains of the polymer are ester groups. In specific embodiments,75% or more of the groups bonded to the amine of the amine side chainsof the polymer are ester groups. In specific embodiments, 90% or more ofthe groups bonded to the amine of the amine side chains of the polymerare ester groups.

In other specific embodiments of formula I, 10%-25% of the groups bondedto the amine of the amine side chains of the polymer are ester groups.In specific embodiments, 25%-50% of the groups bonded to the amine ofthe amine side chains of the polymer are ester groups. In specificembodiments, 50%-75% of the groups bonded to the amine of the amine sidechains of the polymer are ester groups. In specific embodiments, 75%-90%of the groups bonded to the amine of the amine side chains of thepolymer are ester groups. In specific embodiments, 90%-100% of thegroups bonded to the amine of the amine side chains of the polymer areester groups.

In specific embodiments of formula I, R₁₋₄ are all hydrogens. In otherspecific embodiments, R₁₋₄ are independently hydrogens or alkyl groupshaving 1-10 carbon atoms. In other specific embodiments, R₁₋₄ areindependently hydrogens or alkyl groups having 1-6 carbon atoms. Inother specific embodiments, R₁₋₄ are independently hydrogens or alkylgroups having 1-3 carbon atoms. In other specific embodiments, R₁₁₋₁₃are independently alkyl groups having 1-10 carbon atoms. In otherspecific embodiments, R₁₁₋₁₃ are independently alkyl groups having 1-6carbon atoms. In other specific embodiments, R₁₁₋₁₃ are independentlyalkyl groups having 1-3 carbon atoms. In other specific embodiments,R₁₁₋₁₃ are independently methyl or ethyl groups.

In specific embodiments of formula I, R₁₄₋₁₉ are independently alkylgroups having 1-10 carbon atoms. In specific embodiments, R₁₄₋₁₉ areindependently alkyl groups having 1-6 carbon atoms. In specificembodiments, R₁₄₋₁₉ are independently alkyl groups having 1-3 carbonatoms.

In specific embodiments of formula I, R₅₋₁₀ are independently a covalentbond or alkylene chains (CH₂)_(p), where each p is an integer rangingfrom 1-10. In specific embodiments, R₅₋₁₀ are independently a covalentbond or alkylene chains (CH₂)_(p), where each p is an integer rangingfrom 1-6. In specific embodiments, R₅₋₁₀ are independently a covalentbond or alkylene chains (CH₂)_(p), where each p is an integer rangingfrom 1-3.

In specific embodiments of formula I, linker groups A₃, B₃, C₃, D₃ andE₃ are independently alkylene chains (CH₂)_(q) where q is an integerranging from 1-10. In specific embodiments, linker groups A₃, B₃, C₃, D₃and E₃ are independently alkylene chains (CH₂)_(q) where q is an integerranging from 1-6. In specific embodiments, linker groups A₃, B₃, C₃, D₃and E₃ are independently alkylene chains (CH₂)_(q) where p is an integerranging from 1-3. In specific embodiments, linker groups A₃, B₃, C₃, D₃and E₃ are all —CH₂— groups.

In specific embodiments of formula I, Z₁-Z₅ are all covalent bonds andthe backbone of the polymer is not hydrolytically degradable. Inspecific embodiments, the backbone of the polymer is not enzymaticallyor hydrolytically degradable.

In another aspect, the invention relates to multilayer polyelectrolytefilms which are formed from cationic charge dynamic polymers selectedfrom polymers having formula II:

where n+m=N is the number of repeating units in the polymer;each y, independently, is 1, 2 or 3; each x, independently, is aninteger ranging from 1-10;each R¹, each R², each R³ and each R⁴, independently, is selected fromthe group consisting of hydrogen, alkyl groups, alkenyl groups, alkynylgroups, carbocyclic groups, heterocyclic groups, aryl groups, heteroarylgroups, ether groups, all of which may be substituted or unsubstituted.In specific embodiments, each y is the same and each x is the same. Inspecific embodiments, x of the ester groups is different from x on theamide groups. In specific embodiments, each R¹, each R², each R³ andeach R⁴, independently, is selected from the group consisting ofhydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, and benzyl,each of which is optionally substituted.

In specific embodiments, y is 1, x is 1-6, each R¹ and each R⁴,independently, are hydrogen or C1-C3 alkyl, R² are all hydrogen, one R³is hydrogen and two R³ are C1-C3 alkyl. In other specific embodiments,each R¹ is a C1-C3 alkyl. In additional specific embodiments, each R⁴ isa hydrogen. In yet other specific embodiments, each R¹ is a C1-C3 alkyland each R⁴ is a hydrogen.

In specific embodiments of formula II, m is zero. In other embodiments,n is zero. In specific embodiments N=5 to 100,000. More specifically, Ncan range from 20 to 100,000, from 100 to 100,000, from 1,000 to 100,000or from 10,000 to 100,000. In specific embodiments, (n)/(m+n) rangesfrom 0.1 to 1, including 0.05 to 0.25, 0.25 to 0.50, 0.25 to 0.75, 0.75to 1, 0.85 to 1, 0.90 to 1 or 0.95 to 1. In specific embodiments,(n)/(n+m) is 0.50 to 1. In other specific embodiments, (n)/(n+m) is 0.01to 0.5.

In specific embodiments of formula II, 10% or more of the groups bondedas side groups to the polymer are ester groups. In specific embodiments,25% or more of the groups bonded as side groups to the polymer are estergroups. In specific embodiments, 50% or more of the groups bonded asside groups to the polymer are ester groups. In specific embodiments,75% or more of the groups bonded as side groups to the polymer are estergroups. In specific embodiments, 90% or more of the groups bonded asside groups to the polymer are ester groups.

In other specific embodiments of formula II, 10%-25% of the groupsbonded as side groups to the polymer are ester groups. In specificembodiments, 25%-50% of the groups bonded as side groups to the polymerare ester groups. In specific embodiments, 50%-75% of the groups bondedas side groups to the polymer are ester groups. In specific embodiments,75%-90% of the groups bonded as side groups to the polymer are estergroups. In specific embodiments, 90%-100% of the groups bonded as sidegroups to the polymer are ester groups.

The polyelectrolyte films of this invention can provide for release ofanions with separate and/or distinct release profiles. Thepolyelectrolyte films can release the same anion with separate anddistinct release profiles or preferably two or more different anionswith separate and distinct release profiles. More specifically, thepolyelectrolyte films can release two or more different anions eachexhibiting separate and distinct release profiles. In specificembodiments, the anions are nucleic acids, and in particular are nucleicacids which encode one or more polypeptides. In specific embodiments,the nucleic acids are in a form that is capable of expressing one ormore polypeptides. In specific embodiments, the nucleic acids arecomprised in one or more expression cassettes or expression vectors.

In a specific embodiment, the polyelectrolyte film includes at least oneanion/cationic polymer bilayer formed with a first cationic polymer offormula I and at least one such bilayer formed with a second cationicpolymer of formula I.

In another specific embodiment, the polyelectrolyte film includes atleast one anion/cationic polymer bilayer formed with a cationic polymerof formula II. In this embodiment, the film can optionally include acombination of bilayers formed from two or more different cationicpolymers of formula II. In this embodiment, the film can optionallyinclude a combination of bilayers formed from two or more differentanions with the same or different cationic polymers of formula II.

In another specific embodiment, the polyelectrolyte film includes atleast one anion/cationic polymer bilayer formed with a cationic polymerof formula I and at least one such bilayer formed with a cationicpolymer of formula II. In this embodiment, the bilayers formed from thecationic polymer of formula I and formula II can contain the same ordifferent anions.

In a specific embodiment, the polyelectrolyte film includes at least oneanion/cationic polymer bilayer formed with a first cationic polymer offormula I and at least one such bilayer formed with a second cationicpolymer of formula I and at least one such bilayer formed with acationic polymer of formula II. In this embodiment, the bilayers formedfrom the two or more different cationic polymers of formula I and thecationic polymer of formula II can each contain the same or differentanions.

The polyelectrolyte film is preferably generated by layer-by-layerdeposition of the anion(s) and selected cationic polymer(s). Thepolyelectrolyte film comprises a plurality of anion/cationic polymerbilayers. In specific embodiments, the film comprises a plurality ofnucleic acid/cationic polymer bilayers.

The first and second cationic polymers of formula I are structurallydistinct. In specific embodiments, the polyelectrolyte film includes atleast one anion/cationic polymer bilayer formed with a first cationicpolymer of formula I and a first anion and at least one such bilayerformed with a second cationic polymer of formula I and a second anion.

The first and second anions may be the same or different anions. Inspecific embodiments, the anions are nucleic acids. In specificembodiments, the first and second anions are nucleic acids havingdifferent nucleic acid sequences. In specific embodiments, the nucleicacids each encode one or more polypeptides. In specific embodiments, thefirst and second nucleic acids encode first and second polypeptides. Inspecific embodiments, the first and second nucleic acids are comprisedin first and second expression cassettes or expression vectors.

In additional embodiments, the polyelectrolyte film includes two or moreanion/cationic polymer bilayers each of which is formed from a differentcationic polymer of formula I. In additional embodiments, thepolyelectrolyte film includes two or more anion/cationic polymerbilayers each of which is formed from a different cationic polymer offormula I and each of which is formed with a different anion. Inadditional embodiments, the polyelectrolyte film includes two, three ormore nucleic acid/cationic polymer bilayers each of which is formed froma different cationic polymer of formula I and each of which is formedwith a different nucleic acid.

In additional embodiments, the polyelectrolyte film includes two or moreanion/cationic polymer bilayers one of which is formed from a cationicpolymer of formula I and the other of which is formed from a cationicpolymer of formula II. In additional embodiments, the polyelectrolytefilm includes two or more anion/cationic polymer bilayers one of whichis formed from a first anion and a cationic polymer of formula I and oneof which is formed with a second anion and a cationic polymer of formulaII. In specific embodiments, the first and second anions are first andsecond nucleic acids. In specific embodiments, the first and secondnucleic acids are comprises in a first and second expression cassette orvector. In additional embodiments, the polyelectrolyte film can includetwo, three or more anion/cationic polymer bilayers (including nucleicacid/cationic polymer bilayers) each of which is formed from one, two ormore cationic polymers of formula II in combinations with one, two ormore different cationic polymers of formula I wherein each of thebilayers is also formed with a different anion (including differentnucleic acids). In typical embodiments, however a polyelectrolyte filmof the invention will be employed to carry and release one anion, twodifferent anions or three different anions, including one nucleic acid,two different nucleic acids or three different nucleic acids.

In specific embodiments, the polyelectrolyte film is formed on asubstrate. The substrate can be the surface of an implantable medicaldevice from which in certain embodiments, one or two or more differentanion(s) can be delivered to tissues or cells with separate and distinctrelease profiles. In more specific embodiments, the anion(s) in thepolyelectrolyte film on the substrate, including the implantable medicaldevice are nucleic acids and the nucleic acids can be delivered to atissue or cell. In specific embodiments, delivery of nucleic acid totissue or cell results in expression of nucleic acid in the tissue orcell.

Polyelectrolyte assemblies or films of this invention comprise aplurality of polyelectrolyte bilayers wherein at least one bilayercomprises a cationic polymer of formula I or at least one bilayercomprises a cationic polymer of formula II. In certain embodiments, thepolyelectrolyte assembly of the invention includes multipleanion/polycation bilayers, preferably more than two bilayers. Inembodiments containing multiple bilayers, these bilayers mayalternatively differ from each other in their specific anion compositionand/or cationic polymer. Accordingly, respective bilayers mayincorporate one or more anions of different structure. Bilayers maydiffer from each other in their specific polycation makeup as in certainembodiments where differing polycations, including polycations offormula I, formula II or both and optionally polycations other thanthose of formula I or II, which may be degradable and/or non-degradable,may be combined within a single bilayer, or, alternatively, containedwithin distinct bilayers.

In specific embodiments, polyelectrolyte films of this inventioncomprise at least two different bilayers which are formed with at leasttwo different cationic polymers of formula I. In specific embodiments,polyelectrolyte films of this invention comprise a plurality ofanion/cationic polymer bilayers and in at least one and preferably in atleast two such bilayers, the cationic polymer is a polymer of formula I.In specific embodiments, polyelectrolyte films of this inventioncomprise a plurality of anion/cationic polymer bilayers wherein in allsuch bilayers the cationic polymer is a polymer of formula I. Inspecific embodiments, the polyelectrolyte films comprise a plurality offirst bilayers formed from an anion and a first cationic polymer offormula I and a plurality of second bilayers formed from an anion and asecond cationic polymer of formula I. The first and second pluralitiesof bilayers may be formed into a layer configuration in which the firstplurality of bilayers are grouped together and the second plurality ofbilayers are grouped together and the first and second pluralities ofbilayers are optionally separated by one or more intermediate bilayers.

In other specific embodiments, polyelectrolyte films of this inventioncomprise at least two different bilayers which are formed with at acationic polymer of formula I and a cationic polymer of formula II. Inspecific embodiments, polyelectrolyte films of this invention comprise aplurality of anion/cationic polymer bilayers and in at least one andpreferably in at least two such bilayers, the cationic polymer is acationic polymer of formula I or a cationic polymer of formula II. Inspecific embodiments, polyelectrolyte films of this invention comprise aplurality of anion/cationic polymer bilayers wherein in all suchbilayers the cationic polymer is a polymer of formula I or a cationicpolymer of formula II. In specific embodiments, the polyelectrolytefilms comprise a plurality of first bilayers formed from an anion and acationic polymer of formula I and a plurality of second bilayers formedfrom an anion and a cationic polymer of formula II. The first and secondpluralities of bilayers may be formed into a layer configuration inwhich the first plurality of bilayers are grouped together and thesecond plurality of bilayers are grouped together and the first andsecond pluralities of bilayers are optionally separated by one or moreintermediate bilayers.

Each polyelectrolyte assembly of the invention can optionally compriseone or more top protective bilayers and/or one or more base bilayers.One or more base bilayers can be formed between a substrate surface andan anion/polymer cation bilayer where the anion is intended forcontrolled release. A plurality of such base layers may intervenebetween the substrate surface and any anion/cationic polymer bilayers.Base layers, if present, are the bottom most layers in a polyelectrolyteassembly. An intermediate bilayer or a plurality of intermediate layersmay intervene between bilayers or pluralities of bilayers ofanion/cationic polymers where the anion is intended for controlledrelease. One or more top protective bilayers can be positioned as thetop most bilayers in a polyelectrolyte assembly. Intermediate, topprotective and base bilayers can comprise a cationic polymer of formulaI or formula II or both and an anion other than an anion the release ofwhich is intended to be temporally controlled. Intermediate, topprotective and base bilayers can comprise a cationic polymer other thanone of formula I or II, but which is degradable. For example, thecationic polymer of the top, base or intermediate layer may be acationic polymer in which the polymer backbone can degrade, such as apoly(beta-amino0 ester. When the anions to be released from the filmsare one or more nucleic acids, the anion of the intermediate, topprotective and base layers are anions other than nucleic acids which maybe polymeric anions. In specific embodiments, the anions of theintermediate, top protective or base bilayers of the polyelectrolyteassembly are poly(styrene sulfonate).

Generally, a polyelectrolyte film of this invention comprises onebilayer or more than one sequential bilayers for each different anionthat is to be released with a separate and distinct release profile.Each such different one or more sequential bilayers is optionallyseparated from each other different one or more sequential bilayers byone or more intermediate bilayers. In specific embodiments, thepolyelectrolyte film comprises one or more sequential first bilayers andone or more sequential second bilayers and optionally comprises one ormore sequential third bilayers, one or more sequential fourth layers,and one or more sequential fifth bilayers. Each of the different one ormore sequential bilayers is optionally separated from each otherdifferent one or more sequential bilayers by one or more intermediatelayer as noted above.

In specific embodiments, the polyelectrolyte film comprises two, three,four or more different bilayers wherein the different bilayers compriseat least two different cationic polymers of formula I. In other specificembodiments, the polyelectrolyte film comprises two, three, four or moredifferent bilayers wherein the different bilayers comprise at least onecationic polymers of formula I and one cationic polymer of formula II.In general, a bilayer can differ in cationic polymer(s) or in anion(s)present in the bilayer. A given bilayer can comprise two or moredifferent cationic polymers (which may or may not be cationic polymersof formula I or II) including at least one cationic polymer of formula Ior one cationic polymer of formula II. A given bilayer can comprise twoor more different anions which may be different nucleic acids. In apreferred embodiment, a given bilayer contains one cationic polymer offormula I and one anion or a given bilayer contains one cationic polymerof formula II and one anion.

In specific embodiments, the polyelectrolyte assembly can comprise asingle anion a first portion of which is to be released with separate ordistinct release profile compared to a second portion thereof. Thepolyelectrolyte film can comprises two or more anions which are to bereleased with separate, distinct or both separate and distinct releaseprofiles. In specific embodiments, at least one of such anions is anucleic acid. In other embodiments, at least two of such anions aredifferent nucleic acids

In a specific embodiment, the invention provides a polyelectrolyteassembly formed on a substrate. In specific embodiments, the substrateis an implantable medical device. In specific embodiments, wherein thepolyelectrolyte assembly comprises an anion that is a nucleic acid, theimplantable medical device is capable of localized delivery of nucleicacid to a cell. In such an implantable medical device a polyelectrolyteassembly of this invention coats at least a portion of a surface of thedevice. This polyelectrolyte assembly includes at least one nucleicacid/polycation bilayer fabricated by layer-by-layer deposition ofnucleic acid and a polycation of formula I, a polycation of formula IIor both.

A wide range of implantable devices are adaptable for use in the presentinvention including, but not limited to, a stent, a pacemaker, adefibrillator, an artificial joint, a prosthesis, a neurostimulator, aventricular assist device, congestive heart failure device, anindwelling catheter, an insulin pump, an incontinence device, a cochleardevice, or an embolic filter.

Polyelectrolyte assemblies optionally comprise polycations other thanthose of formula I or formula II which are hydrolytically orenzymatically degradable polycations including, but not limited to,poly(beta-amino ester)s, poly(4-hydroxy-L-proline ester),poly[alpha-(4-aminobutyl)-L-glycolic acid], and combinations thereof.

Polyelectrolyte assemblies of this invention optionally comprisepolycations other than those of formula I or formula II which arenon-degradable polycations.

In certain embodiments, the polyelectrolyte assembly of the inventionincludes multiple nucleic acid/polycation bilayers, preferably more thantwo bilayers. In embodiments containing multiple bilayers, thesebilayers may alternatively differ from each other in their specificcomposition of nucleic acid and/or polycation. Accordingly, respectivebilayers may incorporate varied nucleic acids that differ by nucleicacid sequence and those nucleic acids may, in alternative embodiments,be incorporated into one or more expression vectors. Different nucleicacids may encode the same polypeptide, but be under the control ofdifferent regulatory sequences, which affect the level, location ortiming of expression of the polypeptide coding sequences. Differentnucleic acids may encode different polypeptides, but be under thecontrol of the same or similar regulatory sequences such that the level,location or timing of expression of the polypeptide coding sequences isthe same or similar. Similarly, bilayers may differ from each other intheir specific polycation makeup as in certain embodiments wherediffering cationic polymers, including cationic polymers of formula I orformula II and optionally cationic polymers other than those of formulaI or formula II which may be combined within a single bilayer, or,alternatively, contained within distinct bilayers, either with orwithout the presence of non-degradable cationic polymers

In some embodiments, the nucleic acid present in a bilayer encodes apolypeptide such as, for example, endostatin, angiostatin, an inhibitorof vasoactive endothelial growth factor (VEGF), an inhibitor of a signalprotein in a signaling cascade of vascular endothelial growth factor,and inhibitor of basic fibroblast growth factor (bFGF), an inhibitor ofa signal protein in a signaling cascade of bFGF, or combinationsthereof.

In certain embodiments, the polyelectrolyte assembly includes at leasttwo nucleic acids that differ from each other in nucleotide sequence.These respective nucleic acids typically reside in different bilayersand, in carrying out the method, are released from the polyelectrolyteassembly with separate, distinct or both separate and distinct releaseprofiles.

In specific embodiments, in which a polyelectrolyte assembly (amultilayer film) comprises a plurality of first bilayers formed with acationic polymer of formula I and a plurality of second bilayers formedwith a cationic polymer of formula II, the same anions or two or moredifferent anions can be released such that a first selected anion ormixture of anions is released relatively rapidly over a period rangingfrom hours up to about 10 days and a second anion or mixture of anionsis released after a delay or lag period of at least about 20 days.

In another aspect, the present invention encompasses a method ofreleasing two or more anions into a selected environment wherein atleast two of the anions are released from the assembly with separate,distinct or both separate and distinct release profiles. In specificembodiments, the environment is in vivo. In other specific embodiments,the environment is in vitro. Such a method includes steps of contactingthe selected environment with a polyelectrolyte assembly of thisinvention comprising two or more anions. In such method, one or moreanions are released by disruption of one or more bilayers containingsuch one or more anions by decreasing the cationic charge on one or morecationic polymers of formula I, formula II or both.

In a more specific embodiment, the present invention encompasses amethod of delivering two or more nucleic acids into a selectedenvironment comprising one or more cells wherein at least two of thenucleic acids are released from the assembly with separate, distinct orboth release profiles and thus are delivered to the one or more cellswith separate, distinct or both delivery profiles. Such a methodincludes a step of contacting a cell with a polyelectrolyte assembly ofthe invention which comprised two or more nucleic acids. In such method,one or more nucleic acids are released by disruption of one or morebilayers containing such one or more anions by decreasing the cationiccharge on one or more cationic polymers of formula I, formula II orboth. Methods of delivery of nucleic acid according to the invention canbe carried out in the presence of cell culture medium or, alternativelyand more preferably, in the context of a medical device implanted in aliving tissue.

In another aspect, the invention is directed to a method of providing animplantable medical device capable of delivery of two or more anionswherein at least two of the anions are released from the device withseparate, distinct or both separate and distinct release profiles. Inparticular, the two or more anions are two or more nucleic acids. Inparticular, the anions are released into living tissue or cells. Inparticular, the anions are delivered for uptake into one or more cells.In particular, the anions are nucleic acids and the nucleic acids aredelivered to one or more cells in contact with or located at animplantation site of the respective device.

The method includes steps of layer-by-layer depositing of anions andcationic polymer of formula I, formula II or both on a surface of animplantable medical device to provide a polyelectrolyte assembly coatingat least a portion of the implantable medical device. In a specificembodiment, the polyelectrolyte assembly includes at least two differentnucleic acid/cationic polymer bilayers.

The polyelectrolyte assemblies, devices and methods of the presentinvention are particularly advantageous in that they allow for releaseof two or more anions with selected release profiles and/or delivery totissue or cells of two or more anions with selected release profiles. Ina specific embodiment, a device can be coated with a film comprising twoor more nucleic acid sequences, which delivers two or more nucleic acidsto cells or tissues with separate, distinct or both temporal profiles toprovide for transfection of cells of a subject in situ providing for theproduction of therapeutic agents that facilitate a certain therapeuticactivity including, for example, the acceptance of the device by thesubject through the reduction of inflammation associated with implantplacement.

Additional aspects and embodiments of the invention will be apparentupon review of the following non-limiting description, drawings andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a ‘charge-shifting’ polymersynthesized by the conjugate addition of methyl acrylate topoly(allylamine). Gradual hydrolysis of ester-functionalized side chainsintroduces carboxylate functionality and reduces the net charge of thepolymer. Relative changes in net charge shown are exemplary and providedfor illustrative purposes only (see text). Bottom: Polymer 2 is cationicand can be used to fabricate DNA-containing polyelectrolyte multilayers.Time-dependent changes in the net charge of the polymer result inchanges in the nature of the ionic interactions in the multilayers andpromote film erosion and the release of DNA.

FIG. 2 is a graph showing the kinetics of side chain ester hydrolysisfor polymers 2a (Δ), 2b (▴), 2c (□) and 2d (▪) in deuteratedphosphate-buffer (pH=7.4) at 37° C., as determined by ¹H NMRspectroscopy.

FIG. 3 is a plot of film thickness versus the number of polymer/DNAbilayers deposited for films fabricated from plasmid DNA and either PAH(), polymer 2a (Δ), 2b (▴), 2c (□), or 2d (▪) on planar siliconsubstrates.

FIG. 4A is a plot of film erosion vs time for polymer/DNA films eightbilayers thick fabricated from DNA and either PAH (), 2a (Δ), 2b (▴),2c (□), 2d (▪), or polymer 3 (∘) upon incubation in PBS at 37° C.Decreases in film thickness were determined by ellipsometry and areexpressed as a percentage of the original thickness at each time point.

FIG. 4B is a plot of absorbance at 260 nm vs time showing release of DNAfrom films in part A above, fabricated from DNA and either PAH (), 2a(Δ), 2b (▴), 2c (□), 2d (▪), or polymer 3 (∘). Markers representabsorbance values recorded for the incubation buffer; error bars in mostcases are smaller than the symbols used.

FIG. 5 is a plot of the percentage of DNA released vs time for therelease of fluorescently labeled DNA from films having the generalstructure (2c/pEGFP-Cy5)₄(2c/SPS)₂(2a/pDsRed-Cy3)₄ incubated in PBS at37° C. Data points correspond to amounts of pDsRed-Cy3 (Δ) and pEGFP-Cy5(□) in solution determined from solution fluorescence measurements.

FIG. 6 shows representative fluorescence microscopy images showingrelative levels of EGFP (green channel) and RFP (red channel) expressedin COS-7 cells. Cells were transfected with samples of DNA released froma film having the structure (2c/pEGFP)₄(2c/SPS)₂(2a/pDsRed)₄; cells weretransfected by combining released DNA with Lipofectamine 2000 as atransfection agent. The presence, absence, or relative levels of EGFPand RFP observed correspond qualitatively to relative levels of eachplasmid released and collected over each of the following time periods:0-0.5 hrs, 0.5-3 hrs, 3-6 hrs, 6-24 hrs, 24-120 hrs.

FIG. 7 is a graph showing Kinetics of side chain ester hydrolysis forpolymer 5 in phosphate buffer (500 mM, pH=7.2) at 37° C., as determinedby ¹H NMR spectroscopy.

FIG. 8 is a plot of ellipsometric thickness versus the number of polymer5/DNA bilayers deposited on a silicon substrate. Symbols representaverage values and error for multiple independent measurements made onthree different films.

FIG. 9A is a plot of absorbance at 260 nm versus time showing therelease of DNA from films fabricated from polymer 5 (solid diamonds) andpolymer 6 (solid squares). Symbols represent the average and error ofabsorbance values recorded for the incubation buffer.

FIG. 9B is a plot of change in film thickness versus time for filmsfabricated from polymer 5 (solid diamonds) and polymer 6 (solidsquares).

FIGS. 10A, 10B and 10C are schematic illustrations of exemplarypolyelectrolyte assemblies of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an approach for fabrication of multilayer films,i.e. a polyelectrolyte assembly, using ‘charge-shifting’ polymers offormula I or formula II or a combination of such polymers of formula Iand formula II. In contrast to the use of degradable cationic polymers,it is possible to disrupt ionic interactions in polyelectrolyteassemblies of this invention in physiologically relevant media usingcationic polymers designed to undergo gradual reductions in net chargeupon exposure to aqueous media.

In an aspect, in which two or more different cationic polymers offormula I or cationic polymers of each of formula I and II are combined,the polyelectrolyte assemblies (multilayers or films) of this inventionallow release of one or more anions from the assembly exhibitingseparate, distinct or both separate and distinct release profiles. Theinvention is useful for the release of anions from such an assemblywherein at least a portion of the anions are released with a profilethat is distinct and/or separate compared to the release of anotherportion of the anions in the assembly. The first portion and the secondportion of anions may be the same anions or different anions. In aspecific embodiment, the anions are one or more nucleic acids. In aspecific embodiment, the one or more nucleic acids encode one or morepolypeptides and are capable of expressing the encoded polypeptide onrelease from the assembly and introduction of the one or more nucleicacids into tissues or cells.

In an aspect, in which two or more different cationic polymers offormula I or cationic polymers of each of formula I and II are combined,the polyelectrolyte assemblies (multilayers or films) of this inventionallows release of at least two different anions with separate, distinctor both separate and distinct release profiles. The invention isparticularly useful for the release of two or more different anions fromsuch an assembly wherein at least one of the different anions isreleased from the assembly with distinct and/or separate releaseprofiles compared to at least one other anion in the assembly. The firstand the second anions may be the same anions or different anions. In aspecific embodiment the first and second anions are two differentnucleic acids.

In another aspect, in which bilayers are formed from one or morecationic polymers of formula II, the polyelectrolyte assemblies(multilayers or films) of this invention allow long-term release of oneor more anions from the assembly over weeks or months in contrast toshort-term release over hours or days. More specifically, this aspect ofthe invention is useful for the release of one or more anions from theassembly after a delay or lag period of 20 days or more. The inventionis particularly useful for long-term release of one or more nucleicacids from the assembly over weeks or months. More specifically, theinvention is useful for the release of one or more nucleic acids fromthe assembly after a delay or lag period of 20 days or more.

In another aspect, in which bilayers are formed from one or morecationic polymers of formula I and one or more cationic polymers offormula II, the polyelectrolyte assemblies (multilayers or films) ofthis invention allows a combined short-term release of a portion of theanions in the assembly over hours or days and a long-term release ofanother portion of the anions in the assembly over weeks or months. Thefirst portion of anions that are released over a short term (typically10 days or less) may comprise one anion or a mixture of more than oneanions. The second portion of anions that are released long-term(typically 20 days or more) may comprise one anion or a mixture of morethan one anion. The first portion of anions may be the same or differentfrom the second portion of anions. For example, a first anion may bereleased short-term and a different anion may be released long-term.Alternatively, the same anion that was released shot-term may bereleased long-term after a delay or lag period. More specifically, thisaspect of the invention is useful for the release of one or more anionsfrom the assembly for a period up to 10 days and for release of one ormore anions from the assembly after a delay or lag period of 20 days ormore. The invention is particularly useful for controlled short-term andlong-term release of one or more nucleic acids.

Two anions exhibit “distinct” release profiles if the relative amount ofthe two anions released is not constant as a function of time. Twoanions exhibit “separate” release profiles if a portion of one of theanions is released when there is no release of the second anion. In aspecific embodiment, two anions can exhibit “selective separate” releaseif one of the anions is predominantly (50% by weight or more in thepolyelectrolyte assembly) released prior to the release of the secondanion. In a specific embodiment, two anions can exhibit “sequential”release if one of the anions is substantially (90% by weight or more inthe polyelectrolyte assembly) released prior to the release of thesecond anion. In a specific embodiment, two anions can exhibit “distinctsequential” release if one of the anions is approximately completelyreleased (99% by weight or more in the polyelectrolyte assembly) priorto the initiation of release of the second anion. It will be understoodthat two or more different anions can exhibit distinct and/or separateand/or selective separate and/or sequential release and/or distinctsequential release profiles on release from an appropriatepolyelectrolyte assembly of this invention.

It will, however, also be understood that when only a single anion ispresent in the polyelectrolyte assembly, a portion of the anion in agiven assembly can exhibit release that is distinct and/or separateand/or selective separate and/or sequential and/or distinct sequentialcompared to another portion of the same anion in that assembly.Differences in the release profile of a single anion can be assessed bytagging or labeling sub-portions of the anion to be released, forexample, employing isotopic or radiolabeling.

In view of the descriptions herein regarding anion release profiles fromrepresentative cationic polymers of formulas I and II, one of ordinaryskill in the art can prepare polyelectrolyte assemblies to achievedesired distinct, separate, distinct and separate, long-term orcombinations of long-term and short-term release profiles by combininganion/cationic polymer bilayers formed from selected polymers offormulas I and II. The choice of cationic polymer is generally madebased on the structure of the polymer as described particularly in theexamples herein.

In specific embodiments, the invention provides multilayerpolyelectrolyte films comprising two or more different anion/cationicbilayers which provide for distinct and/or separate and/or selectiveseparate and/or sequential release and/or distinct sequential releaseprofiles of anions therein.

The present invention relates to multilayer polyelectrolyte films whichare formed from at least two different cationic charge dynamic polymers,also called charge shifting polymers, selected from cationic polymershaving formula I:

where:n, m, l, k and i are zero or integers where n+m+l+k+i=N, the totalnumber of repeat units in the polymer;A₁₋₃, B₁₋₃, C₁₋₃, D₁₋₃ and E₁₋₃ are linkers which may be the same oreach may be different;Z₁-Z₅ are covalent bonds or degradable bonds;R₁₋₄ and R₁₁₋₁₉, independently, can be hydrogen, or alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups with theexception that R₁₋₄ are not esters; andR₅-R₁₀ are linkers or covalent bonds which may be the same or each maybe different. Variables in formula I are further described above.

In specific embodiments, cationic polymers of formula IA are useful inthis invention:

where variables are as defined above. The number of repeating units inthe polymer N is n+m+

. In specific embodiments, (m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1.

In specific embodiments, cationic polymers of formula IB are useful inthis invention:

where each r is an integer ranging from 1-10 and other variables are asdefined above. The number of repeating units in the polymer N is n+m+

. In specific embodiments, (m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1. In specific embodiments, each r is 1, 2 or 3. Inspecific embodiments, all r have the same value.

In specific embodiments, cationic polymers of formula IC are useful inthis invention:

where variables are as defined above. The number of repeating units inthe polymer N is n+m+

. In specific embodiments, (m+l)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to0.25, 0.25 to 0.50, 0.25 to 0.75, or 0.75 to 1. In specific embodiments,each r independently is 1, 2 or 3 and each p independently is 1, 2 or 3.In specific embodiments, all r have the same value. In specificembodiments, all p have the same value.

In specific embodiments, cationic polymers of formula ID are useful inthis invention:

where variables are as defined above. The number of repeating units inthe polymer N is m+

+k+i. In specific embodiments, (m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1. In specific embodiments, k is 0, k is less than 0.01or k is less than 0.1. In specific embodiments, each r is 1, 2 or 3. Inspecific embodiments, all r are the same. In specific embodiments, R₃and R₄ are hydrogens.

In specific embodiments, cationic polymer of formula IE are useful inthis invention:

where variables are as defined above. The number of repeating units inthe polymer N is n+m+

. In specific embodiments, (m+

)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to0.75, or 0.75 to 1. In specific embodiments, each r independently is 1,2 or 3 and each p independently is 1, 2 or 3. In specific embodiments,all r have the same value. In specific embodiments, all p have the samevalue. In specific embodiments, R₃ and R₄ are hydrogens.

In specific embodiments of formulas IA-IE all of R₁₁-R₁₉ are alkylgroups having 1-8, 1-6 or 1-3 carbon atoms.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise two or more different cationic polymers of any of formulasIA-IE where each different cationic polymer (m+

)/N is a different value.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a cationic polymer of any offormulas I, or IA-IE where (m+

)/N is 0.01 to 0.50, 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, or 0.25 to0.50 and a second bilayer comprising a cationic polymer of any offormulas I, or IA-IE where (m+

)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1, wherein the first andsecond cationic polymers have different values of (m+

)/N.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a cationic polymer of any offormulas I, or IA-IE where (m+

)/N is 0.01 to 0.25, 0.01 to 0.1, 0.05 to 0.2, or 0.1 to 0.25, and asecond bilayer comprising a cationic polymer of any of formulas I, orIA-IE where (m+l)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1.0.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a cationic polymer of any offormulas I, or IA-IE where (m+

)/N is 0.01 to 0.25, a second bilayer comprising a cationic polymer ofany of formulas i, or IA-IE where (m+

)/N is 0.50 to 0.75 and a third bilayer comprising a cationic polymer ofany of formulas I, or IA-IE where (m+

)/N is 0.80 to 1.0.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise one more intermediate bilayers each comprising a cationicpolymer of any of formulas I or IA-IE.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise one or more intermediate bilayers each comprising a cationicpolymer of any of formulas I, or IA-IE where (m+

)/N is 0.01 to 0.25, 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, or 0.25 to0.5. In specific embodiments, polyelectrolyte assemblies of thisinvention comprise one or more intermediate bilayers each comprising acationic polymer of any of formulas I, or IA-IE where (m+

)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1.0.

In specific embodiments, cationic polymers of formula II include thoseof formula IIA, IIB, IIC, IID, IIE or IIF which are all useful in thisinvention:

where variable are as defined above. In specific embodiments of formulasIA-IF, all R³ are alkyl groups and particularly are C1-C3 alkyl groups.In other embodiments, all R³ are methyl. In other embodiments offormulas IA-IF, all R¹ are hydrogen or alkyl. In other embodiments, allR¹ are C1-C3 alkyl. In other embodiments, all R¹ are methyl. In specificembodiments, all x are the same. In other embodiments all y are thesame. In specific embodiments, each y is 2. In specific embodiments,each x is 2, 3 or 4.

In specific embodiments of formulas IIA-IIF, m is zero. In otherembodiments of formulas IIA-IIF, n is zero. In specific embodiments offormulas IIA-IIF, N=5 to 100,000. More specifically, N can range from 20to 100,000, from 100 to 100,000, from 1,000 to 100,000 or from 10,000 to100,000. In specific embodiments of formulas IIA-IIF, (n)/(m+n) rangesfrom 0.1 to 1, including 0.05 to 0.25, 0.25 to 0.50, 0.25 to 0.75, 0.75to 1, 0.85 to 1, 0.90 to 1 or 0.95 to 1. In specific embodiments offormulas IIA-IIF, (n)/(n+m) is 0.50 to 1. In other specific embodimentsof formulas IIA-IIF, (n)/(n+m) is 0.01 to 0.5.

In specific embodiments of formulas IIA-IIF, 10% or more of the groupsbonded as side groups to the polymer are ester groups. In specificembodiments, 25% or more of the groups bonded as side groups to thepolymer are ester groups. In specific embodiments, 50% or more of thegroups bonded as side groups to the polymer are ester groups. Inspecific embodiments, 75% or more of the groups bonded as side groups tothe polymer are ester groups. In specific embodiments, 90% or more ofthe groups bonded as side groups to the polymer are ester groups.

In other specific embodiments of formulas IIA-IIF, 10%-25% of the groupsbonded as side groups to the polymer are ester groups. In specificembodiments, 25%-50% of the groups bonded as side groups to the polymerare ester groups. In specific embodiments, 50%-75% of the groups bondedas side groups to the polymer are ester groups. In specific embodiments,75%-90% of the groups bonded as side groups to the polymer are estergroups. In specific embodiments, 90%-100% of the groups bonded as sidegroups to the polymer are ester groups.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise one or two or more different cationic polymers of any offormulas II or IIA-IIF. In specific embodiments, polyelectrolyteassemblies of this invention comprise two or more different cationicpolymers of any of formulas II or IIA-IIF, where each different cationicpolymer has (n)/(n+m) that is different.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas II,or IIA-IIF where (n)/(n=m) is 0.1 to 1, 0.25 to 1, 0.5 to 1, or 0.75 to1 and a second bilayer comprising a polycation of any of formulas II, orIIA-IIF where (n)/(n=m) is 0.01 to 0.25, 0.1 to 0.25, 0.01 to 0.1 or0.05 to 0.25.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise at least a first bilayer comprising a polycation of any offormulas I, or IA-IE and at least a second bilayer comprising apolycation of any of formulas II, or IIA-IIF.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise at least a first bilayer comprising a polycation of any offormulas I, or IA-IE and at least a second bilayer comprising apolycation of any of formulas II, or IIA-IIF wherein m is zero or(n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise at least a first bilayer comprising a polycation of any offormulas I, or IA-IE wherein and at least a second bilayer comprising apolycation of any of formulas II, or IIA-IIF wherein m is zero or(n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas I,or IA-IE where (m+l)/N is 0.01 to 0.25, or a first bilayer comprising apolycation of any of formulas I, or IA-IE where (m+l)/N is 0.50 to 0.75or a first bilayer comprising a polycation of any of formulas I, orIA-IE where (m+l)/N is 0.80 to 1.0 in combination with a second bilayerof a polycation of any of formulas II or IIA-IIF where m is 0 or(n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas I,or IA-IE where (m+

)/N is 0.01 to 0.25, a second bilayer comprising a polycation of any offormulas I, or IA-IE where (m+

)/N is 0.50 to 1 in combination with a third bilayer of a polycation ofany of formulas II or IIA-IIF where m is 0 or (n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas I,or IA-IE where (m+l)/N is 0.50 to 1, a second bilayer comprising apolycation of any of formulas I, or IA-IE where (m+

)/N is 0.80 to 1.0 in combination with a third bilayer of a polycationof any of formulas II or IIA-IIF where m is 0 or (n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas I,or IA-IE where (m+

)/N is 0.80 to 1.0 and a second bilayer comprising a polycation of anyof formulas II or IIA-IIF where m is 0 or (n)/(n+m) is 0.5 to 1.

In specific embodiments, polyelectrolyte assemblies of this inventioncomprise a first bilayer comprising a polycation of any of formulas I,or IA-IE where n, m, k and i are all zero and a second bilayercomprising a polycation of any of formulas II or IIA-IIF where m is 0.

In specific embodiments, polyelectrolyte assemblies of this inventionfurther comprise one more intermediate bilayers each comprising acationic polymer of any of formulas II or IIA-IIF.

In specific embodiments, polyelectrolyte assemblies of this inventionfurther comprise one or more intermediate bilayers each comprising acationic polymer of any of formulas I, or IIA-IIF where m is 0, or where(n)/(n+m) ranges from 0.5 to 1.0. In specific embodiments,polyelectrolyte assemblies of this invention comprise one or moreintermediate bilayers each comprising a cationic polymer of any offormulas II, or IIA-IIF where (n)/(n+m) is 0.1 to 0.5.

In specific embodiments, polyelectrolyte assemblies of this inventionfurther comprise one or more intermediate bilayers each comprising acationic polymer other than a polymer of formulas I or II, but which isa degradable polymer and which in particular is a cationic polymer thepolymer backbone of which is hydrolytically or enzymatically degradable.

The number of bilayers in polyelectrolyte assemblies of this inventionis not particularly limited. The number of bilayers can for example be1-10, 1-20, 1-100, 1-500, or 1-1000. In specific embodiments, 75% to100% of the bilayers in the polyelectrolyte assembly can be those formedby one or more polymers of formula II. In other specific embodiments,75% to 100% of the bilayers in the polyelectrolyte assembly can be thoseformed by one or more polymers of formula I. In other specificembodiments, 50% to 90% of the bilayers in the polyelectrolyte assemblycan be those formed by one or more polymers of formula II, and 10% to50% of the bilayers in the polyelectrolyte assembly can be those formedby a one or more polymers of formula I. In other specific embodiments,50% to 90% of the bilayers in the polyelectrolyte assembly can be thoseformed by one or more polymers of formula I, and 10% to 50% of thebilayers in the polyelectrolyte assembly can be those formed by a one ormore polymers of formula II. In other specific embodiments, 50% to 99%of the bilayers in the polyelectrolyte assembly can be those formed byone or more polymers of formula II, and 1% to 50% of the bilayers in thepolyelectrolyte assembly can be those formed by a one or more polymersof formula I.

In other specific embodiments, 50% to 99% of the bilayers in thepolyelectrolyte assembly can be those formed by one or more polymers offormula I, and 1% to 50% of the bilayers in the polyelectrolyte assemblycan be those formed by a one or more polymers of formula II.

FIGS. 10A-C illustrate exemplary polyelectrolyte assemblies of thisinvention comprising one or more cationic polymers of formulas I, IA,IB, IC, ID, IE, II, IIA, IIB, IIC, IID, IIE, IIF or combinationsthereof.

In the assembly (10) of FIG. 1A, a plurality of sequential firstbilayers (2) and a plurality of second sequential bilayers (3) areoptionally separated by one or more than one intermediate bilayer (4).The assembly optionally comprises one or more base bilayers (5) formedon the substrate (9). The assembly optionally comprises one or more topprotective bilayers (6). The number of first bilayers and secondbilayers can be the same or different. There can be more first bilayersthan second bilayers or there can be fewer first bilayers than secondbilayers. It will be apparent that the assembly of FIG. 10A can containadditional pluralities of bilayers which may be pluralities of the firstor second bilayers or a plurality of one or more different bilayers,e.g., a third, fourth, fifith or other bilayer. Each such plurality ofbilayers is optionally separated by one or more intermediate bilayers.Assemblies as in FIG. 10A can, for example, comprise a total of four ormore different bilayers. In specific embodiments, assemblies of FIG. 10Acomprise 1000, 500 or 200 or fewer bilayers in addition to any basebilayers. In specific embodiments, assemblies of FIG. 10A comprise 100,50 or 20 or fewer bilayers in addition to any base bilayers. In specificembodiments, assemblies of FIG. 10A comprise 10 or fewer bilayers inaddition to any base bilayers.

The assembly (10) of FIG. 10B, comprises a plurality of sequential firstbilayers (2a), a second plurality of first bilayers (2b) and a pluralityof second sequential bilayers (3). Each plurality of bilayers isoptionally separated by one or more than one intermediate bilayers (4aand 4b). The assembly optionally comprises one or more base bilayers (5)formed on the substrate. (9). The assembly optionally comprises one ormore top protective bilayers (6). The assembly can contain additionalpluralities of first bilayers and/or additional pluralities of secondbilayers. The number of first bilayers and second bilayers in theassembly can be the same or different. There can be more first bilayersthan second bilayers or there can be fewer first bilayers than secondbilayers. It will be apparent that the assembly of FIG. 10B can containadditional pluralities of bilayers which may be pluralities of the firstor second bilayers or a plurality of one or more different bilayers,e.g., a third, fourth, fifth or other bilayer. Each such plurality ofbilayers is optionally separated by one or more intermediate bilayers.The number of bilayers in the first plurality of first bilayers and thenumber of bilayers in any additional pluralities of first bilayers canbe the same or different. The number of bilayers in the first pluralityof second bilayers and the number of bilayers in any additionalpluralities of second bilayers can be the same or different. Assembliesas in FIG. 10B can, for example, comprise a total of 6 or more bilayers.In specific embodiments, assemblies of FIG. 10B comprise 1000, 500, 200or fewer bilayers in addition to any base bilayers. In specificembodiments, assemblies of FIG. 10B comprise 100, 50, 20 or fewerbilayers in addition to any base bilayers. In specific embodiments,assemblies of FIG. 10B comprise 10 or fewer bilayers in addition to anybase bilayers. In an assembly of FIG. 10B it will be recognized that theorder of the pluralities of bilayers in the assembly can be changed. Forexample, first and second pluralities of first bilayers can be layeredsequentially, separated by one or more intermediate bilayers, and firstand second pluralities of second bilayers can be layered sequentiallyseparated by one or more intermediate bilayers.

The assembly (10) of FIG. 1C comprises one or a plurality of (two ormore) sequential first bilayers (2), one or a plurality of secondbilayers (3) and one or a plurality of third sequential bilayers (7).Each plurality of bilayers is optionally separated by one or more thanone intermediate bilayers (not shown). The assembly optionally comprisesone or more base bilayers (5) formed on the substrate (9). The assemblyoptionally comprises one or more top protective bilayers (6). The numberof first bilayers, second bilayers and third bilayers in the assemblycan be the same or different. There can be more first bilayers thansecond bilayers or there can be fewer first bilayers than secondbilayers. It will be apparent that the assembly of FIG. 10C can containadditional single bilayers or pluralities of bilayers which may befirst, second, third or additional bilayers, e.g., a fourth, fifth orother bilayer. Each such plurality of bilayers is optionally separatedby one or more intermediate bilayers. Assemblies as in FIG. 10C cancomprise a total of three or more bilayers. In specific embodiments,assemblies of FIG. 10C comprise 1000, 500, 200 or fewer bilayers inaddition to any base bilayers. In specific embodiments, assemblies ofFIG. 10C comprise 100, 50, 20 or fewer bilayers in addition to any basebilayers. In specific embodiments, assemblies of FIG. 10C comprise 10 orfewer bilayers in addition to any base bilayers.

Bilayers in any of FIGS. 1A-C are different if they comprise a differentpolycation composition or a different anion composition. Differentbilayers include those formed using different cationic polymers ordifferent mixtures of cationic polymers. Different bilayers includethose comprising different anions or different mixtures of anions. In aspecific embodiment, different bilayers can contain the same anion ormixture of anions which are present in the different bilayers atdifferent concentrations or amounts.

In a specific embodiment, a polyelectrolyte assembly as in FIG. 10A isdesigned for short-term release of a first anion and long-term releaseof a second anion. The anions may be first and second nucleic acids. Inthe assembly, upper bilayers (2) contain the anion to be releasedshort-term (hours or days or 10 days or less) and lower bilayers (3)contain the anion to be released long-term (20 days or more, weeks, ormonths). Bilayers 2 are preferably formed with one or more cationicpolymers of formula I wherein (m+l)/N is 0.5 or more and bilayers 3 arepreferably formed with a cationic polymer of formula II, particularlywhere (n)/(n+m) is 0.5 or more and more specifically where m is 0. Theassembly optionally further comprises one or more base, top orintermediate layers between the short-term and long-term releasebilayers. In specific embodiments, the first bilayers 2 and secondbilayers 3 are sequential and contain 1-1000 (or 1-500, 1-200, 1-200,1-50, 1-20 or 1-10) bilayers and are optionally separated by 1-100 (or1-50, 1-20 or 1-10) intermediate bilayers. In specific embodiments, theassembly comprises 1-100 (or 1-50, 1-20, or 1-10) base bilayers and/or1-100 (or 1-50, 1-20, or 1-10) top protective bilayers.

In specific embodiments, the invention provides polyelectrolyteassemblies comprising 1-1000 (or 1-500, 1-200, 1-100, 1-50 or 1-20)first bilayers formed from a first cationic polymer of any of formulasI, or IA-IE where (m+l)/N is 0.01 to 0.50, 0.01 to 0.1, 0.05 to 0.2, 0.1to 0.25, or 0.25 to 0.50 and 1-1000 (or 1-500, 1-200, 1-100, 1-50 or1-20) second bilayers formed from a second polycation of any of formulasIA-IE where (m+

)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1, wherein the first andsecond polycations have different values of (m+l)/N. In specificembodiments, the first bilayers and second bilayers are sequential andare optionally separated by 1-100 (or 1-50, 1-20 or 1-10) intermediatebilayers. In specific embodiments, the assembly comprises 1-100 (or1-50, 1-20, or 1-10) base bilayers and/or 1-100 (or 1-50, 1-20, or 1-10)top protective bilayers. In specific embodiments, the first and secondbilayers comprise different anions or different mixtures of anions. Inspecific embodiments, the first and second bilayers comprise differentnucleic acids or different mixtures of nucleic acids. In specificembodiments, the first and second bilayers comprise different nucleicacids carried on one or more vectors. In specific embodiments, the firstand second bilayers comprise different nucleic acids carried on one ormore expression vectors. In specific embodiments, the different nucleicacids have different sequences. In specific embodiments, the first andsecond bilayers comprise different nucleic acids which encode one ormore polypeptides. In specific embodiments, the first and secondbilayers comprise different nucleic acids each of which encodes adifferent one or more polypeptides. In specific embodiments, theassembly comprises 1-1000 (or 1-500, 1-200, 1-100, 1-50, 1-20 or 1-10)first bilayers and 1-1000 (or 1-500, 1-200, 1-100, 1-50, 1-20 or 1-10)second bilayers. In specific embodiments, the assembly comprises 1-10first bilayers and 1-10 second bilayers. In specific embodiments, theassembly comprises 1-10 first bilayers and 1-10 second bilayersseparated by 1-10 intermediate bilayers.

Dynamic charge state cationic polymers are polymers designed to havecationic charge densities that decrease by removal of removablefunctional groups from the polymers. In specific embodiments, theremovable functional group is a hydrolysable group, such as a pendantester which is converted on hydrolysis to a pendant —COO⁻ (anionicgroup). For some polymers herein, the ester bond will generally bereadily hydrolysable, whereas the amide bond is not readilyhydrolysable.

The polymers of the present invention may have any desired molecularweight, such as from 1,000 to 100,000 grams/mole, or from about 2,000 to50,000 grams/mole. The dynamic charge state cationic polymers of thisinvention can be associated with a ligand facilitating the delivery ofthe polymer to a specific target, such as a target cell.

The cationic polymers of this invention can also be part of a copolymer,which can be composed of any other polymers, for example a polymer suchas PEG or PEO which are commonly used to give stability toward proteinadsorption. The cationic polymers of the invention are generallycationic, but different functional groups attached to the polymer canrender the polymer zwitterionic. To impart a cationic charge to thepolymer, the attached functional groups can be positively charged. Thecationic polymers of the invention may also be capable of bufferingchanges in pH which results from the make-up of the polymer backboneand/or the attached functional groups. Thus, the invention furtherrelates to polyelectrolyte assemblies which comprise one or morecopolymer comprising a cationic polymer of formula I or formula II.

Certain cationic polymers of this invention carry positive charge onpolymer side chains. Dependent upon the specific structure of thecationic polymer, hydrolysis of side chain groups, such as esters,results in the formation of negatively charged species on the sidechains and an overall decrease in positive charge of the polymer. Inspecific embodiments, the polymer backbones of the cationic polymers ofthis invention do not carry charge. In specific embodiments, the polymerbackbones of the cationic polymers of this invention are nothydrolytically or enzymatically degradable.

The present dynamic charge state cationic polymers may benon-immunogenic, non-toxic or both non-immunogenic and non-toxic. In thepresent polymers, the polymeric backbone can be degradable ornondegradable. The present polymers do not require that the degradationof the backbone occur at the same time as the shift in cationic charge.One skilled in the art will recognize that the measure of degradabilitywill be commensurate with the environmental conditions and desiredproperties for any particular application for the present polymers.

As one non-limiting example, for biomedical uses of the presentpolymers, the present invention contemplates polymers that degrade in adesired time frame (from an hour to a week to a month to a year) underphysiological conditions typically found in the body or in a cell orcell compartment [e.g., pH ranges from about 5.0 (endosomal/lysosomal)to 7.4 (extracellular and cytosol), a temperature of about 37° C. and anionic strength of a typical physiological solution (generally around130-150 mM NaCl, for example)]. In the present invention, thedegradability of the polymer can be measured by a variety of methods,including, but not limited to, GPC (gel permeation chromatography).

The present invention also provides cationic polymers complexed with oneor more anionic molecules thereby forming an interpolyelectrolytecomplex.

Suitable anions of the invention may be naturally occurring, synthetic,or both. In some embodiments, suitable examples of anions includenucleic acids, such as RNA, DNA, and analogs thereof. In otherembodiments, the anion is a synthetic polyanion. In still otherembodiments, the anions of the invention are nucleic acids, such as RNA,DNA, or analogs thereof, and a synthetic polyanion. When the anion is anucleic acid, the nucleic acid can have the sequence of a nucleic acidmolecule of interest or its complement. As such, the nucleic acid canencode for a protein or a functional fragment thereof or be useful inantisense treatment or RNA interference. In some embodiments, thenucleic acid is a plasmid. In other embodiments, the anionic molecule oragent may be a therapeutic molecule, diagnostic molecule, peptide, orcarbohydrate, for example a macromolecular carbohydrate such as heparin.

The following are terms used in the present application:

The term “alkyl” as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and thirty (more typically between 1-22) carbonatoms by removal of a single hydrogen atom. In some embodiments, alkylgroups have from 1 to 12, from 1 to 8 carbon atoms, from 1 to 6 or 1 to3 carbon atoms. Examples of alkyl radicals include, but are not limitedto, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.A “cycloalkyl” group is a cyclic alkyl group typically containing from 3to 8 ring members such as, but not limited to, a cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl group.

The term “alkoxy” as used herein refers to an alkyl group, as previouslydefined, attached to the parent molecular moiety through an oxygen atom.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy groups.

The term “alkenyl” denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. Alkenyl groups typically can have 1-22 carbonatoms and include, for example, ethenyl, propenyl, butenyl,l-methyl-2-buten-l-yl, and the like. Alkenyl groups include those havingfrom 2-12 carbon atoms, those having 2-8, and those having 2-6 carbonatoms.

The term “alkynyl” as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Alkynyl groups can typically have1-22 carbon atoms. Representative alkynyl groups include ethynyl,2-propynyl (propargyl), l-propynyl, and the like. Alkynyl groups includethose having from 2-12 carbon atoms, those having 2-8, and those having2-6 carbon atoms.

Alkyl, alkenyl and alkynyl groups can be optionally substituted withgroups including alkoxy, thioalkoxy, amino, alkylamino, dialkylamino,trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro,carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide groups.

The term “aryl” as used herein refers to carbocyclic ring systems havingat least one aromatic ring including, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, and indenyl groups, and the like.Aryl groups can be unsubstituted or substituted with substituentsselected from the group consisting of branched and unbranched alkyl,alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino,dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto,nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. Inaddition, substituted aryl groups include tetrafluorophenyl andpentafluorophenyl.

The term carbocyclic is used generally herein to refer to groupscontaining one or more carbon rings. The groups may be aromatic or arylgroups. Rings may contain 3-10 carbon atoms and one, two or three doublebonds or a triple bond. These groups may include single rings of 3 to 8atoms in size and bi- and tri-cyclic ring systems which may includearomatic six membered aryl or aromatic groups fused to a non-aromaticring.

The terms “heterocyclic” and “heterocyclyl”, are used broadly herein torefer to an aromatic, partially unsaturated or fully saturated 3- to10-membered ring system, which includes single rings of 3 to 8 atoms insize and bi- and tri-cyclic ring systems which may include aromatic sixmembered aryl or aromatic heterocyclic groups fused to a non-aromaticring. These heterocyclic and heterocyclyl rings and groups include thosehaving from one to three heteroatoms independently selected from oxygen,sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms mayoptionally be oxidized and the nitrogen heteroatom may optionally bequaternary.

The terms “aromatic heterocyclic” or “heteroaryl” as used herein, referto a cyclic aromatic radical having from five to 12 ring atoms of whichone ring atom is selected from sulfur, oxygen, and nitrogen; zero, one,or two ring atoms are additional heteroatoms independently selected fromsulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon,the radical being joined to the rest of the molecule via any of the ringatoms. The term includes heteroaromatic rings fused to aryl ring or tocarbocylic rings. Examples of such aromatic heterocyclyl groups include,but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl groups,and the like.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3-methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine,4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine-,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term “hydrocarbon”, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. The hydrocarbon may be unsaturated, saturated, branched,unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

The terms “substituted”, whether preceded by the term “optionally” ornot, and “substituent”, as used herein, refer to the ability, asappreciated by one skilled in this art, to change one functional groupfor another functional group provided that the valency of all atoms ismaintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents may also be further substituted (e.g., anaryl group substituent may be further substituted. For example, a nonlimiting example is an aryl group that may be further substituted with,for example, a fluorine group at one or more position.

As used herein, “biodegradable” compounds are those that, whenintroduced into cells, are broken down by the cellular machinery or byhydrolysis into components that the cells can either reuse or disposeof, in some cases without significant toxic effect on the cells (e.g.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain embodiments,the chemical reactions relied upon to break down the biodegradablecompounds are uncatalyzed.

A “labile bond” is a covalent bond that is capable of being selectivelybroken. That is, a labile bond may be broken in the presence of othercovalent bonds without the breakage of other covalent bonds. Forexample, a disulfide bond is capable of being broken in the presence ofthiols without cleavage of any other bonds, such as carbon-carbon,carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also bepresent in the molecule. “Labile” also means cleavable.

A “labile linkage” is a chemical compound that contains a labile bondand provides a link or spacer between two other groups. The groups thatare linked may be chosen from compounds such as biologically activecompounds, membrane active compounds, compounds that inhibit membraneactivity, functional reactive groups, monomers, and cell targetingsignals. The spacer group may contain chemical moieties chosen from agroup that includes alkanes, alkenes, esters, ethers, glycerol, amide,saccharides, polysaccharides, and heteroatoms such as oxygen, sulfur, ornitrogen. The spacer may be electronically neutral, may bear a positiveor negative charge, or may bear both positive and negative charges withan overall charge of neutral, positive or negative.

In general, the “effective amount” of an active agent refers to theamount necessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. The polyelectrolyteassemblies of this invention can be employed to deliver an effectiveamount of one or more active agents which are anions and particularlywhich are nucleic acids.

As used herein, “peptide”, means peptides of any length and includesproteins. The terms “polypeptide” and “oligopeptide” are used hereinwithout any particular intended size limitation, unless a particularsize is otherwise stated. The only limitation to the peptide or proteindrug which may be utilized is one of functionality. The terms “protein”and “peptide” may be used interchangeably. Peptide may refer to anindividual peptide or a collection of peptides. peptides preferablycontain only natural amino acids, although non-natural amino acids(i.e., compounds that do not occur in nature but that can beincorporated into a polypeptide chain; see, for example,http://www.cco.caltech.edu/.about.da-dgrplUnnatstruct.gif, whichdisplays structures of non-natural amino acids that have beensuccessfully incorporated into functional ion channels) and/or aminoacid analogs as are known in the art may alternatively be employed.Also, one or more of the amino acids in an peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. In some embodiments, the modifications of the peptidelead to a more stable peptide (e.g., greater half-life in vivo). Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc. Anions of this invention include anionicpolypeptides, proteins and/or peptides.

As used herein, “administering”, and similar terms means delivering thecomposition to the individual being treated. In some instances thecomposition is capable of being circulated systemically where thecomposition binds to a target cell and is taken up by endocytosis. Inspecific embodiments of this invention, polyelectrolyte assemblies canbe employed to administer or deliver two or more anions to anindividual.

The present methods may be carried out by performing any of the stepsdescribed herein, either alone or in various combinations. The presentcompounds may also have any or all of the components described herein.One skilled in the art will recognize that all embodiments of thepresent invention are capable of use with all other embodiments of theinvention described herein. Additionally, one skilled in the art willrealize that the present invention also encompasses variations of thepresent methods and compositions that specifically exclude one or moreof the steps, components or groups described herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

Accordingly, for all purposes, the present invention encompasses notonly the main group, but also the main group absent one or more of thegroup members. The present invention also envisages the explicitexclusion of one or more of any of the group members in the claimedinvention.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. A number of specific groups of variabledefinitions have been described herein. It is intended that allcombinations and subcombinations of the specific groups of variabledefinitions are individually included in this disclosure. Accordingly,for all purposes, the present invention encompasses not only the maingroup, but also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

Compounds described herein may exist in one or more isomeric forms,e.g., structural or optical isomers. When a compound is described hereinsuch that a particular isomer, enantiomer or diastereomer of thecompound is not specified, for example, in a formula or in a chemicalname, that description is intended to include each isomers andenantiomer (e.g., cis/trans isomers, R/S enantiomers) of the compounddescribed individual or in any combination. Additionally, unlessotherwise specified, all isotopic variants of compounds disclosed hereinare intended to be encompassed by the disclosure. For example, it willbe understood that any one or more hydrogens in a molecule disclosed canbe replaced with deuterium or tritium. Isotopic variants of a moleculeare generally useful as standards in assays for the molecule and inchemical and biological research related to the molecule or its use.Isotopic variants, including those carrying radioisotopes, may also beuseful in diagnostic assays and in therapeutics. Methods for making suchisotopic variants are known in the art. Specific names of compounds areintended to be exemplary, as it is known that one of ordinary skill inthe art can name the same compounds differently.

Molecules disclosed herein may contain one or more ionizable groups[groups from which a proton can be removed (e.g., —COOH) or added (e.g.,amines) or which can be quaternized (e.g., amines)]. All possible ionicforms of such molecules and salts thereof are intended to be includedindividually in the disclosure herein. With regard to salts of thecompounds herein, one of ordinary skill in the art can select from amonga wide variety of available counterions those that are appropriate forpreparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt. Every formulation or combination of components described orexemplified herein can be used to practice the invention, unlessotherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. The broad termcomprising is intended to encompass the narrower consisting essentiallyof and the even narrower consisting of. Thus, in any recitation hereinof a phrase “comprising one or more claim element” (e.g., “comprising Aand B), the phrase is intended to encompass the narrower, for example,“consisting essentially of A and B” and “consisting of A and B.” Thus,the broader word “comprising” is intended to provide specific support ineach use herein for either “consisting essentially of” or “consistingof.” The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, catalysts, reagents, synthetic methods, purification methods,analytical methods, and assay methods, other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisinvention. The terms and expressions which have been employed are usedas terms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byexamples, preferred embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. Some references provided herein are incorporated byreference to provide details concerning sources of starting materials;alternative starting materials, reagents, methods of synthesis,purification methods, and methods of analysis; as well as additionaluses of the invention.

Unless otherwise specified, “a” or “an” means “one or more”.

THE EXAMPLES Example 1 Multilayered Films Assembled from Charge-ShiftingCationic Polymers Providing Separate and/or Distinct Release Profiles ofDNA Constructs

In general, approaches to the design of ‘charge-shifting’ polymers havetaken one of two basic routes: (i) the attachment of amine-functionalside chains to polymer backbones through cleavable linkages, or (ii) theconjugate addition of ester-functionalized ‘charge-shifting’ side chainsto the backbones of cationic polymers. [X. H. Liu, J. W. Yang, A. D.Miller, E. A. Nack, D. M. Lynn, Macromolecules 2005, 38, 7907.]

Polymer 1 is exemplary of this second design approach (ii); gradualreductions in the net charge of this polymer can be made to occur uponhydrolysis of ester-functionalized side chains and the introduction ofanionic charge (Eq 1; full protonation of amine functionality is shownfor illustrative purposes).^([24])

This polymer can promote both self-assembly and time-dependentdisassembly with DNA in solution in ways that can be understood in termsof side chain hydrolysis and subsequent changes in the net charges ofthe polymer. This approach also permits tunable control over the natureof electrostatic interactions with DNA by control over the number ofcharge-shifting side chains added to the polymer.

This example demonstrates that this approach to the disruption of ionicinteractions in polyelectrolyte assemblies can be exploited to exertcontrol over the time-dependent stability of polyelectrolyte multilayersin aqueous environments. Recently, it has been reported that‘charge-shifting’ cationic polymers designed using polymers havingamine-functional side chains attached through hydrolyzable linkages canbe used to fabricate multilayers. [J. T. Zhang, D. M. Lynn, Adv Mater2007, 19, 4218; B. G. De Geest, R. E. Vandenbroucke, A. M. Guenther, G.B. Sukhorukov, W. E. Hennink, N. N. Sanders, J. Demeester, S. C. DeSmedt, Adv Mater 2006, 18, 1005.] De Geest et al. in particular reportedthe use of this approach to fabricate multilayered microcapsulesdesigned for intracellular delivery.

In the context of designing films that provide tunable control over filmdisassembly, the approach used to design polymer 1 can provide practicaladvantages relative to the approaches noted above (which require thesynthesis of specialized monomers) because this approach is (i) modularand (ii) it can be used to introduce ‘charge-shifting’ side chains to abroad range of commercially available polyamines. [Liu et al., 2005,supra]

The addition of ester-functionalized ‘charge-shifting’ side chains topoly(allylamine hydrochloride) (PAH) (polymer 2) can be used to providecontrol over the erosion of DNA-containing films and design multilayeredfilms that orchestrate the release of multiple different DNA constructswith separate and distinct release profiles.

The methyl ester-functionalized polymer 2 was synthesized by theconjugate addition of PAH to methyl acrylate using a procedure similarto that described previously for the synthesis of polymer 1. [Liu etal., 2005, supra] Treatment of PAH with an excess of methyl acrylateresulted in the exhaustive functionalization of PAH (i.e., polymer 2;n=m=0), as determined by ¹H NMR spectroscopy. To investigate theinfluence of polymer structure on film growth and behavior, wesynthesized four derivatives of polymer 2 having approximately 100%,75%, 50%, and 25% substitution (referred to hereafter as polymers 2a,2b, 2c, and 2d) by varying the amount of methyl acrylate added. PAHcontains primary amine functionality that can participate in up to twoconjugate addition reactions with methyl acrylate. As a result, thestructures of polymers 2b, 2c, and 2d (each substituted at <100%)consist of mixtures of repeat units that are either exhaustivelyalkylated, partially alkylated, or non-alkylated (as shown in FIG. 1).The extents of substitution of polymers 2b-d are reported here aspercentages relative to the number of side chains that would be presentin an exhaustively substituted polymer (e.g., polymer 2a, n=m=0).

The side chain methyl esters of polymers 2a-d hydrolyze to unmaskanionic carboxylate functionality when these materials are incubated inphysiologically relevant media (e.g., FIG. 1, top). Characterization ofester hydrolysis in deuterated phosphate-buffered saline (PBS, pH=7.4)at 37° C. revealed differences in the rates of hydrolysis for these fourmaterials (e.g., half-lives ranging from ˜4.5 days for polymer 2a to ˜1day or less for polymers 2b-d; see FIG. 2). Characterization of theresulting acid-functionalized materials by FTIR spectroscopydemonstrated that side chain hydrolysis occurred without the formationof amide crosslinks between the amine functionality and esterfunctionality in these materials.

A series of experiments was conducted to determine whetherester-functionalized polymers 2a-d could be used to fabricatepolyelectrolyte multilayers using a plasmid DNA construct (pEGFP-N1)encoding enhanced green fluorescent protein (EGFP) and an alternatedipping procedure similar to that used in our past studies to fabricatefilms using hydrolytically degradable cationic polymers. [J. Zhang, L.S. Chua, D. M. Lynn, Langmuir 2004, 20, 8015; C. M. Jewell, J. Zhang, N.J. Fredin, D. M. Lynn, J Control Release 2005, 106, 214; C. M. Jewell,J. Zhang, N. J. Fredin, M. R. Wolff, T. A. Hacker, D. M. Lynn,Biomacromolecules 2006, 7, 2483.]

For these and all other experiments described below, films werefabricated on planar silicon substrates to permit characterization offilm growth and erosion using ellipsometry.

FIG. 3 shows a plot of optical film thickness versus the number ofpolyamine/DNA layers (referred to hereafter as ‘bilayers’) deposited forfilms fabricated using either polymers 2a-d or unsubstituted PAH.Inspection of these data reveals that the optical thicknesses of allfilms increased in a manner that was linear or roughly linear withrespect to the number of polymer/DNA bilayers deposited. Furtherinspection, however, reveals large differences in rates of film growthand final film thicknesses. For example, films 8 bilayers thickfabricated using polymers 2a and 2b were ˜110 nm thick, whereas filmsfabricated using polymers 2c and 2d were only ˜45 nm thick after thedeposition of 8 bilayers. The thicknesses of films fabricated using lesssubstituted polymers 2c and 2d (which should have a higher percentage ofunsubstituted amine functionality and, thus, greater PAH character) are,in general, closer in thickness to films fabricated using PAH (˜28 nm).These observations, when combined, indicate that alkylation of theprimary amines of PAH may influence the ability of these polymers toform electrostatic interactions with DNA (for example, by creating moresterically hindered secondary or tertiary amines) or lead to differencesin the ionization or solution conformations of the polymers in ways thatinfluence the thickness of each adsorbed layer.

The stability of films fabricated from polymers 2a-d were characterizedin physiologically relevant media to determine whether differences inthe structures of these ester-functionalized polymers could be exploitedto provide control over rates of film erosion and the release of DNA(e.g., FIG. 1, bottom). FIG. 4A shows a plot of decreases in filmthickness for DNA-containing films fabricated using either PAH orpolymers 2a-d upon incubation in PBS at 37° C. Inspection of the data inFIG. 4A reveals that the thickness of films fabricated usingunsubstituted PAH (closed circles) does not decrease significantly forup to 250 hours. These results demonstrate that DNA-containingmultilayers fabricated from PAH are stable for at least 10 days underthese conditions, and provide a baseline from which to characterizetime-dependent changes in the stability of films fabricated frompolymers 2a-d.

The data in FIG. 4A reveal large differences in the stabilities of filmsfabricated from polymers 2a-d that correlate to differences in theamount of ester-functionalized side chains incorporated into thesematerials. Films fabricated from 100%-substituted polymer 2a (opentriangles) decreased in film thickness very rapidly, and essentiallycompletely, within the first hour of incubation in PBS. The thicknessesof films fabricated from 75%-substituted polymer 2b also decreasedrapidly, although not as completely, during the first hour (e.g., an˜80% decrease within the first hour), with the remainder of the filmeroding more slowly over an additional two day period.

These results demonstrate that films fabricated from these two polymersare unstable and erode rapidly upon incubation in PBS. The smalldifferences in erosion profile noted above correlate, in general, withdifferences in the number of side chains incorporated into thesepolymers. Film erosion occurs sufficiently rapidly in these cases,however, that it is difficult to interpret this behavior solely in termsof side chain hydrolysis or the potential ‘charge-shifting’ nature ofthese ester-functionalized materials. For example, rapid decreases infilm thickness are also consistent with film dissolution processes thatcould occur upon the immersion of these ionically crosslinked assembliesin solutions of high ionic strength (e.g., PBS).

To probe the nature of the film disassembly processes further, weconducted an additional series of experiments using films fabricatedfrom DNA and amide-substituted polymer 3.

Polymer 3 is an analog of polymer 2a with dimethylamide-functionalizedside chains (synthesized by the conjugate addition ofN,N-dimethylacrylamide to PAH; see below) that do not hydrolyze readilyunder the conditions used here. Inspection of the data in FIG. 4Areveals that films fabricated using polymer 3 (open circles) are stableand do not decrease in thickness for up to 10 days under theseconditions. These data, when combined with those above, provide supportfor the view that the ester functionality in polymer 2 plays animportant role in governing the stability (or instability) of thesematerials in PBS.

The remaining data in FIG. 4A correspond to films fabricated frompolymers 2c and 2d and demonstrate further that rates of film erosionare influenced significantly by the number of ester side chainsincorporated into the polymer. Films fabricated from 25%-substitutedpolymer 2d (closed squares) were stable and did not decrease inthickness substantially for over 10 days when incubated in PBS buffer.However, films fabricated using 50%-substituted polymer 2c (opensquares) decreased in thickness gradually over a period of 10 days underthese conditions.

The physical erosion of these films also results in the surface-mediatedrelease of plasmid DNA into solution. FIG. 4B shows a plot of solutionabsorbance (at 260 nm, the absorbance maximum of DNA) versus timemeasured during the film erosion experiments described above. Thedifferences in the DNA release profiles shown in FIG. 4B are consistentwith the differences in the erosion profiles shown in FIG. 4A, anddemonstrate that it is possible to control the rates at which DNA isreleased from film-coated surfaces by changing the structure of thepolyamines used to fabricate the films. The differences in the finalsolution absorbance values arising from films fabricated from polymers2a and 2b and films fabricated from less-substituted polymer 2ccorrelate directly to differences in the amounts of DNA in these films,and correlate to differences in the initial thicknesses of these films(see FIG. 3). These differences in film erosion and DNA release profilescan be exploited to design films with architectures that permit controlover the release of two DNA constructs with separate and distinctrelease profiles.

Several recent reports have demonstrated that layer-by-layer methods ofassembly can be used to fabricate polyelectrolyte multilayers composedof multiple different layers of multiple different polyelectrolytes. [SN. Jessel, M. Oulad-Abdelghani, F. Meyer, P. Lavalle, Y. Haikel, P.Schaaf, J. C. Voegel, Proc Natl Acad Sci USA 2006, 103, 8618; T. Dubas,T. R. Farhat, J. B. Schlenoff, J Am Chem Soc 2001, 123, 5368. J. Cho, F.Caruso, Macromolecules 2003, 36, 2845. A. J. Nolte, M. F. Rubner, R. E.Cohen, Langmuir 2004, 20, 3304. K. C. Wood, H. F. Chuang, R. D. Batten,D. M. Lynn, P. T. Hammond, Proc Natl Acad Sci USA 2006, 103, 10207.]

Jessel et al. demonstrated recently that this general approach could beused to fabricate DNA-containing multilayers that provide control overthe order in which two different DNA constructs were expressed byattached cells (e.g., by depositing two different plasmid DNA constructsat different depths within an enzymatically degradable film). Zhang etal. also demonstrated that hydrolytically degradable polyamines could beused to fabricate films that provide control over the release of twoplasmid constructs into solution. [J. T. Zhang, S. I. Montanez, C. M.Jewell, D. M. Lynn, Langmuir 2007, 23, 11139.]

This approach permitted measures of control over the relative orderswith which two plasmid constructs were released (e.g., by controllingthe relative orders with which they were incorporated into the films),but it was not possible to fabricate films that provided largedifferences in individual release profiles. For example, it was notpossible to fabricate films capable of regulating the release of twodifferent DNA constructs with separate and mutually exclusive releaseprofiles (that is, films for which one DNA construct could be releasedlargely before the onset of the release of a second DNA construct). Wehave demonstrated this type of control for release of DNA in exemplarymultilayers formed employing polymers 2a and 2c.

Films were fabricated using both the pEGFP-N1 plasmid described aboveand a second plasmid construct (pDsRed-N1) encoding red fluorescentprotein (RFP). In the experiments described below, we used filmsfabricated from polymers 2a and 2c and either (i) plasmid DNAfluorescently labeled with Cy5 or Cy3 fluorescent dyes (denotedpEGFP-Cy5 and pDsRed-Cy3; used to permit characterization of the releaseprofiles of each plasmid independently using fluorimetry), or (ii)unlabeled plasmid DNA (to permit characterization of gene expression incell-based assays). Films used in these experiments were fabricatedlayer-by-layer to contain four bottommost layers containing polymer 2c(which released DNA slowly in the above experiments) and four topmostlayers containing polymer 2a (which released DNA rapidly in the aboveexperiments).

Additionally two bilayers fabricated from polymer 2c and sodiumpoly(styrene sulfonate) (SPS) were deposited as intermediate layersbetween the plasmid-containing layers of these films. Films having thisgeneral structure are denoted hereafter in the following manner:(2c/plasmid₁)₄(2c/SPS)₂(2a/plasmid₂)₄ (see also the schematicillustration in FIG. 6).

FIG. 5 shows the results of an experiment conducted using a film havingthe structure (2c/pEGFP-Cy5)₄(2c/SPS)₂(2a/pDsRed-Cy3)₄. Inspection ofthese data reveals that the pDsRed-Cy3 plasmid, deposited in the topmostlayers of the film, is released rapidly and completely within the first30 min of incubation in PBS (open triangles). In contrast, the pEGFP-Cy5plasmid, deposited in the bottommost layers of the film, is releasedmore slowly over a period of 48 hours (open squares). The relative orderin which these two plasmids are released is consistent with the order inwhich they were deposited, and the relative rates at which they arereleased are consistent with the behaviors of polymer 2a (rapid release)and polymer 2c (slow release) observed in the experiments describedabove. Reversing the order in which the two different plasmids weredeposited during fabrication [i.e., using films having the generalstructure (2c/pDsRed-Cy3)₄(2c/SPS)₂(2a/pEGFP-Cy5)₄] resulted in areversal of the order in which the DNA constructs were released.

Additional consideration of the data in FIG. 5 reveals that the releaseprofiles for each DNA construct are distinct and almost completelynon-overlapping (e.g., only ˜15% of the pEGFP-Cy5 plasmid is releasedduring the time required for all of the pDsRed-Cy3 plasmid to bereleased). These results are believed to arise from the largedifferences in the release profiles that can be achieved using polymers2a and 2c.

These results also indicate that a relatively low level of physicalinterpenetration may exist among the layers in the topmost andbottommost portions of these films. Additional delay in the onset of therelease of the plasmid located in the bottommost layers of these filmscan be obtained by manipulating the number or structure of theintermediate layers deposited between the DNA-containinglayers.^([17,34])

We conducted another set of experiments using films having the structure(2c/pEGFP)₄(2c/SPS)₂(2a/pDsRed)₄ (that is, films identical to thosedescribed above, but fabricated using plasmid that was not fluorescentlylabeled) to characterize the functional integrity of released DNA anddetermine whether the differences in the release profiles shown in FIG.5 could also be observed as differences in EGFP and RFP expressionprofiles in cells. FIG. 6 shows a series of fluorescence micrographs ofCOS-7 cells 48 hours after treatment with samples of plasmid DNAcollected at five predetermined time points during the erosion of thesefilms.

Inspection of the data in the left column of FIG. 6 (red fluorescencechannel) reveals high levels of red fluorescence in cells treated withsamples of DNA collected after 30 min of incubation. Further inspectionreveals little red fluorescence in cells treated with DNA collected atsubsequent time points. These data demonstrate that the pDsRed plasmidreleased from these films is released in a form that remainstranscriptionally active, and they provide an additional indication thatessentially all of the pDsRed located in the topmost layers of the filmis released within the first 30 min of incubation. The right column ofFIG. 6 (green fluorescence channel) shows images of the same cells shownin the left column. These images demonstrate that significant levels ofEGFP expression are not observed in cells treated with samples of DNAcollected at 30 min, but that the number of cells expressing EGFPincreases throughout the remainder of the experiment. These temporaldifferences in the expression of EGFP and RFP are consistent with theresults shown in FIG. 5 and demonstrate that it is possible to exploitthe structures and properties of polymer 2 to design films that permitcontrol over the release of two different DNA constructs with releaseprofiles that are distinct and essentially non-overlapping.

This work provides an approach to the fabrication of ultrathinpolyelectrolyte multilayers that provides temporal control over therelease of two different DNA constructs from surfaces. The addition ofester-functionalized side chains to poly(allylamine) provides controlover the stability of DNA-containing multilayers in aqueousenvironments. By control over the number of ester-functionalized sidechains added to the polymer, it is possible to design films that releaseDNA rapidly, slowly, or that are stable and do not release DNA uponincubation in physiologically relevant media. These differences in filmerosion can be exploited to design multilayers with architectures thatprovide control over the release of two or more different plasmidconstructs with distinct and largely non-overlapping release profiles.Such control has been difficult to achieve using conventional methodsfor the incorporation of DNA into thin films and coatings. We and othershave demonstrated in past reports that polyelectrolyte multilayersfabricated from DNA can be used to promote localized andsurface-mediated cell transfection.^([23]) In this context, the approachreported here contributes to the development of thin films and coatingscapable of regulating the localized release of well-defined quantitiesof multiple different DNA constructs (or other agents) of interest in abroad range of fundamental and applied contexts

Materials. Test grade n-type silicon wafers were purchased from Si-Tech,Inc. (Topsfield, Mass.). Poly(allylamine hydrochloride) (PAH, MW≈60,000)was obtained from Alfa Aesar Organics (Ward Hill, Pa.). Sodiumpoly(styrene sulfonate) (SPS, MW=70,000), methyl acrylate, andN,N-dimethylacrylamide were obtained from Aldrich Chemical Co.(Milwaukee, Wis.). Plasmid DNA [pEGFP-N1 or pDsRed2-N1 (4.7 kb, >95%supercoiled)] was purchased from Elim Biopharmaceuticals, Inc. (SanFrancisco, Calif.). Cy3 and Cy5 Label-IT nucleic acid labeling kits werepurchased from Mirus (Madison, Wis.). All commercial materials were usedas received without further purification unless otherwise noted.Deionized water (18 MΩ) was used for washing steps and to prepare allpolymer solutions. PBS buffer was prepared by diluting commerciallyavailable concentrate (EM Science) and adjusting the pH to 7.4 with 1.0M HCl or NaOH. All buffers and polymer solutions were filtered through a0.2-μm membrane syringe filter prior to use unless otherwise noted.

General Considerations. ¹H and ¹³C nuclear magnetic resonance (NMR)spectra were recorded on Bruker AC+ 300 (300.135 MHz) and Varian UNITY500 (499.896 MHz) spectrometers. Chemical shift values are given in ppmand are referenced with respect to residual protons from solvent.Attenuated total reflectance infrared spectroscopy data were collectedon a Bruker TENSOR 27 FTIR instrument (Billerica, Mass.) outfitted withan ATR transmission cell from PIKE Technologies (Madison, Wis.). Siliconsubstrates (e.g., 0.5×2.0 cm²) used for the fabrication of multilayeredfilms were cleaned with methylene chloride, ethanol, methanol, anddeionized water, and dried under a stream of filtered compressed air.Surfaces were then activated by etching with oxygen plasma for 5 min(Plasma Etch, Carson City, Nev.) prior to film deposition. The opticalthicknesses of films deposited on silicon substrates were determinedusing a Gaertner LSE ellipsometer (632.8 nm, incident angle=70°). Datawere processed using the Gaertner Ellipsometer Measurement Program.Relative thicknesses were calculated assuming an average refractiveindex of 1.577 for the multilayered films. Thicknesses were determinedin at least five different standardized locations on each substrate andare presented as an average (with standard deviation) for each film. Allfilms were dried under a stream of filtered compressed air prior tomeasurement.

UV-visible absorbance values for phosphate-buffered saline (PBS)solutions used to determine film release kinetics were recorded on aBeckman Coulter DU520 UV-vis spectrophotometer (Fullerton, Calif.).Absorbance values were recorded at a wavelength of 260 nm (theabsorbance maximum of DNA). Fluorescence measurements of solutions usedto erode multilayered films fabricated from DNA labeled with Cy3 and Cy5fluorescent dyes were made using a Fluoromax-3 fluorimeter (Jobin Yvon,Edison, N.J.). Fluorescence microscopy images used to evaluate theexpression of enhanced green fluorescent protein (EGFP) or redfluorescent protein (RFP) in cell transfection experiments were recordedusing an Olympus IX70 microscope and were analyzed using the Metavueversion 4.6 software package (Universal Imaging Corporation). Imageacquisition settings were identical for all samples, using an exposuretime of 200 ms, a gain of +0.25, and a binning of two. Data were storedin single channel, 12-bit TIF format. Additional image processing waslimited to false coloring and scaling.

Synthesis of Ester-Functionalized PAH (Polymer 2). The conjugateaddition of PAH to methyl acrylate was performed using a protocolsimilar to that reported previously for the synthesis ofester-functionalized linear poly(ethylene imine) (LPEI) [24]. PAH (550mg) was dissolved in methanol (˜5 wt % in methanol) and 1.1 mL of asodium methoxide solution (35 wt % in methanol) was added. The resultingreaction mixture was stirred for 4 hr at 45° C., precipitated NaCl wasremoved by filtration, and methyl acrylate was added. The amount ofmethyl acrylate added was varied (e.g., from 0.5 to 2.2 equivalentsrelative to the molar amount of amine functionality in PAH) to achievedesired mole percent substitutions. Reaction mixtures were stirred atroom temperature for two hours (for polymers synthesized at lowacrylate/amine ratios) or up to 48 hours (for polymers synthesized athigher acrylate/amine ratios). Reactions requiring longer reaction timeswere monitored to prevent the formation of amide crosslinks resultingfrom potential reactions between amines and the ester functionality ofmethyl acrylate using attenuated total reflectance infraredspectroscopy. One equivalent of HCl was added to the reaction mixture,and the resulting reaction product was concentrated by rotaryevaporation. The final product was dissolved in a mixture ofdichloromethane and methanol (v/v=9:1) and precipitated into hexanes.The isolated material was dried under vacuum to yield the desiredproduct as a white solid in near quantitative yield. Representative ¹HNMR data for a polymer with 100% substitution: (D₂O) δ (ppm)=1.6 (br,2H); 2.1 (br, 1H); 2.8-3.3 (br, 8H); 3.5 (br, 2H), 3.72 (s, 6H).

Synthesis of Amide-Functionalized PAH. The conjugate addition of PAH toN,N-dimethylacrylamide was performed using a protocol similar to thatreported previously for the synthesis of amide-functionalized LPEI [24]and conducted in analogy to the synthesis of polymer 2 above.Representative ¹H NMR data for a polymer with 100% substitution: (D₂O) δ(ppm)=1.6 (br, 2H); 2.1 (br, 1H); 2.8-3.3 (br m, 20H); 3.4 (br, 2H).

Characterization of Side Chain Ester Hydrolysis. ¹H NMR experiments usedto characterize the kinetics of ester hydrolysis forester-functionalized PAH in physiologically relevant media wereconducted in the following general manner. Ester-functionalized polymer(˜10 mg) was dissolved in deuterated PBS buffer (0.6 mL, pH˜7.4),3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (˜3 mg) was addedas an internal standard, and the resulting solution was placed in aglass NMR tube. The NMR tube was placed in a 37° C. incubator andremoved periodically for analysis by ¹H NMR spectroscopy. Thedisappearance of the methyl ester resonance at 3.72 ppm was monitoredand integrated versus the trimethylsilyl protons of the internalstandard.

Preparation of Polyelectrolyte Solutions. Solutions of Cationic Polymersused for dipping (10 mM with respect to the MW of the polymer repeatunit) were prepared in 18 MΩ water and pH was adjusted to ˜5 using 1NNaOH. Solutions of SPS (20 mM with respect to the MW of the polymerrepeat unit) were prepared in 18 MΩ water. DNA solutions (1 mg/mL) usedfor the deposition of polymer/DNA layers were prepared in sodium acetatebuffer (100 mM, pH=5) and were not filtered prior to use.

Fabrication of Multilayered Films. Multilayered films were fabricated onplanar silicon substrates using an alternating dipping procedureaccording to the following general protocol: (1) Substrates weresubmerged in a solution of polycation for 5 min, (2) substrates wereremoved and immersed in an initial water bath for 1 min followed by asecond water bath for 1 min, (3) substrates were submerged in a solutionof polyanion for 5 min, and (4) substrates were rinsed in the mannerdescribed above. This cycle was repeated until the desired number ofpolycation/polyanion layer pairs (typically eight) had been deposited.For experiments designed to characterize film growth profiles byellipsometry, films were dried after every two cycles of the aboveprocedure using filtered compressed air prior to measurement. Films tobe used in erosion and release experiments were either used immediatelyafter fabrication or dried under a stream of filtered compressed air andstored in a vacuum desiccator until use. All films were fabricated atambient room temperature.

Characterization of Film Erosion and Release Kinetics. Experimentsdesigned to investigate film erosion and release kinetics were performedin the following general manner: Film-coated substrates were placed in aplastic UV-transparent cuvette and 1.0 mL of PBS (pH=7.4, 137 mM NaCl)was added to cover the film-coated portion of the substrate. The sampleswere incubated at 37° C. and removed at predetermined intervals forcharacterization by ellipsometry. Films were rinsed under deionizedwater and dried under a stream of filtered compressed air prior tomeasurement. Values of optical film thickness were determined in atleast four different predetermined locations on the substrate byellipsometry and the samples were returned immediately to the buffersolution. For experiments designed to monitor the concentration of DNAin solution, UV absorbance readings were made using the solution used toincubate the sample (at 260 nm, the absorbance maximum of DNA). Forexperiments in which fluorescently labeled DNA was used, changes in theconcentration of DNA in solution were monitored by fluorimetry. Forrelease experiments designed to produce samples of DNA suitable for usein cell transfection experiments, erosion experiments were conducted asdescribed above with the following exceptions: at each predeterminedtime interval substrates were removed from the buffer, placed into a newcuvete containing fresh PBS, and the original DNA-containing solutionwas stored for use in transfection experiments.

Cell Transfection Assays. COS-7 cells were grown in 96-well plates at aninitial seeding density of 12,000 cells/well in 200 mL of growth medium(90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum,penicillin 100 units/mL, streptomycin 100 mg/mL). Cells were grown for24 h, at which time 50 mL of a Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) and plasmid mixture was added directly to the cells according tothe general protocol provided by the manufacturer. The Lipofectamine2000/plasmid transfection milieu was prepared by mixing 25 mL of theplasmid solution collected at each time point during release experiments(arbitrary concentrations but constant volumes) with 25 mL of dilutedLipofectamine 2000 reagent (24 mL stock diluted into 976 mL of water).Fluorescence images were taken after 48 h using an Olympus IX70microscope and analyzed using the Metavue version 4.6 software package(Universal Imaging Corporation).

Example 2 Preparation of Polyelectrolyte Multilayers for ExtendedLong-Term Release of Nucleic Acid

Side-chain functionalized polymer 5 was synthesized by the reaction of3-dimethylamino-1-propanol with poly (2-vinyl-4,4-dimethyl azlactone)(4, Mn ˜50,000; Scheme 1). This general approach permits conjugation oftertiary amine-functionalized side chains to a poly(acrylamide) backbonethrough a hydrolysable ester bond. Polymer 5 is a weak polyelectrolyte;it is soluble in aqueous media and behaves as a cationic polymer byvirtue of protonation of pendant tertiary amines. As illustrated inScheme 1 hydrolysis of the ester bonds in the side chains of polymer 5leads to gradual loss of amine functionality and the introduction ofanionic carboxylate functionality. Thus, polymer 5 is capable oftransforming gradually from a polymer that is completely positivelycharged to a polymer that is completely negatively charged upon completeside chain hydrolysis.

¹H NMR spectroscopy was used to characterize the loss of esterfunctionality in solutions of polymer 5 as a function of time uponincubation in phosphate buffer (pH=7.2; 37° C.). The results of theseexperiments demonstrate that side chain hydrolysis occurs slowly inphysiologically relevant media (t½ ˜200 days; see FIG. 7). Incomplete orpartial hydrolysis of the side chains in polymer 5 would lead to apolymer containing both cationic and anionic side chains and that, ingeneral, the overall net charge of these polymers would depend uponadditional environmental factors such as pH and ionic strength.

Polymer 5 was used to fabricate multilayered films using a plasmid DNAconstruct (pEGFP-N1) encoding enhanced green fluorescent protein (EGFP).All films used in these initial studies were deposited on planar siliconsubstrates to permit characterization of film thicknesses and growthprofiles using ellipsometry FIG. 8 shows a plot of the optical thicknessof films versus the number of polymer 5/DNA layers (hereafter referredto as ‘bilayers’) deposited. Film thickness increased in a nonlinearmanner for the first three bilayers and then, subsequently, as a linearfunction of the number of bilayers deposited, resulting in films ˜100 nmthick after the deposition of 8 bilayers.

On the basis of these optical measurements, the average thickness of apolymer 2/DNA bilayer in these films was ˜12.5 nm. Characterization ofthese films by atomic force microscopy (AFM) revealed the surfaces ofthese assemblies to be rough (RRMS ˜47 nm; data not shown). Thethicknesses and surface morphologies of these films are similar to thosereported in past studies for the assembly of multilayered films usingplasmid DNA and a variety of other cationic polymers. [J. Blacklock, H.Handa, D. Soundara Manickam, G. Mao, A. Mukhopadhyay, D. Oupicky,Biomaterials 2007, 28, 117; J. Chen, S. Huang, W. Lin, R. Zhuo, Small2007, 3, 636; N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir 2005, 21,5803.]

Additional experiments were performed to determine whether assembliesfabricated from polymer 5 and plasmid DNA could erode and release DNAwhen incubated in aqueous media. FIG. 9A (closed diamonds) shows a plotof solution absorbance (at 260 nm, the absorbance maximum of DNA) as afunction of time for a polymer 5/DNA film ˜80 nm thick incubated in PBSat 37° C.

These data demonstrate that DNA is released into solution over a periodof 90 days. On the basis of these absorbance data, the amount of DNAincorporated into a film ˜80 nm thick was estimated to be ˜4.8 μg/cm².Further inspection of this release profile reveals the presence of a lagphase of ˜25 days prior to the release of measurable amounts of DNA intosolution. This behaviour contrasts significantly to that ofpolyamine/DNA films fabricated from hydrolytically or enzymaticallydegradable polyamines, for which DNA is generally observed to bereleased immediately upon exposure of film to aqueous environments orenzymes (and often with an initial burst of DNA release). [J. Zhang, L.S. Chua, D. M. Lynn, Langmuir 2004, 20, 8015; K. F. Ren, J. Ji, J. C.Shen, Biomaterials 2006, 27, 1152; C. M. Jewell, J. Zhang, N. J. Fredin,M. R. Wolff, T. A. Hacker, D. M. Lynn, Biomacromolecules 2006, 7, 2483.]The presence of a lag phase in this current system provides insight intopossible molecular level processes that may contribute to the extendedrelease profiles of these materials.

FIG. 9B (closed diamonds) shows a plot of film thickness versus timecorresponding to the erosion profile for the film shown in FIG. 9A. Filmthickness does not decrease significantly over the first ˜25 days. Thisperiod of apparent film stability corresponds closely to the lag phasein the DNA release profile shown in FIG. 9A. Film thickness decreases ina nearly linear manner upon further incubation, corresponding to theperiod of time over which DNA is observed to be released into solution.Characterization of the surfaces of these films during erosion by AFMrevealed changes in surface morphologies from films that were initiallyrough (RRMS ˜40 nm; as described above) to surfaces that were smooth anduniform (RRMS ˜3 nm) over a period of ˜20 days. This behaviour variesconsiderably from the behaviour of multilayered films fabricated usingplasmid DNA and hydrolytically degradable poly(β-amino ester)s, whichundergo dramatic changes in nanometer-scale surface structure uponincubation in PBS. [N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir 2005,21, 5803; N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir 2007, 23, 2273.]

These results demonstrate that polymer 5 can be used to fabricateultrathin films that erode and release plasmid DNA over long periods oftime. This behavior is believed to result from the gradual hydrolysis ofthe side chains in polymer 5 which is supported, in part, by thesolution-phase side-chain hydrolysis experiments discussed above.

Polymer 6 was synthesized by the reaction of3-dimethylamino-1-propylamine with polymer 4. Polymer 6 has a molecularweight, polydispersity, and chemical structure that is identical to thatof polymer 5, with the exception that the tertiary amine functionalityof the side chain is linked to the backbone of the polymer through anamide linkage that does not hydrolyze readily in physiologicallyrelevant media.

Amide-functionalized polymer 6 was used to fabricate DNA containingfilms on silicon substrates using a procedure identical to thatdescribed above for polymer 5. The growth profiles of polymer 6/DNAfilms were similar that of polymer 5/DNA films (data not shown).However, striking differences were observed in stability of filmsfabricated from polymers 5 and 6 when films were incubated in PBS.

As shown in FIGS. 9A and 9B (closed squares), films fabricated frompolymer 6 did not decrease in thickness or release measurable amounts ofDNA into solution for periods of up to 90 days. The results of theseexperiments demonstrate that replacement of the ester functionality inpolymer 5 with amide functionality leads to assemblies that do not erodeor release DNA under otherwise identical conditions. These resultsprovide strong support that the erosion and release of DNA from filmsfabricated from polymer 5 results from the hydrolysis of the side chainsof polymer 5, and not from other factors (such as changes in pH or ionicstrength) that could arise during the incubation of these assemblies.

The hydrolysis of the side chains of polymer 5 should result in a changein the net charge of the polymer and, as a result, a change in thenature of electrostatic interactions within an ionically crosslinkedfilm. The results indicate that such changes in the strength of theseionic interactions are sufficient to disrupt these films and promote therelease of DNA. The erosion and release of DNA from films fabricatedfrom polymer 5 occurs over periods of time ˜55 times longer than filmsfabricated from hydrolytically or enzymatically degradable polymers(under otherwise similar conditions). [J. Zhang, L. S. Chua, D. M. Lynn,Langmuir 2004, 20, 8015; K. F. Ren, J. Ji, J. C. Shen, Biomaterials2006, 27, 1152; C. M. Jewell, J. Zhang, N. J. Fredin, M. R. Wolff, T. A.Hacker, D. M. Lynn, Biomacromolecules 2006, 7, 2483.]

One possible explanation for these differences is that the hydrolysis ofthe side chains in polymer 5 occurs slowly in PBS (as noted above).However, we also considered fundamental differences in the structures ofthese cationic polymers that could lead to such large differences infilm behaviour. For example, for films fabricated using hydrolyticallyor enzymatically degradable cationic polymers, mechanisms of filmerosion and DNA release involve polymer chain backbone scission. Inassemblies fabricated from these degradable polymers, the hydrolysis ofa single bond in the backbone of a polymer chain can result in adramatic change in the molecular weight of the polymer and, as a result,a significant reduction of the stability of a film. By contrast, thebackbone of polymer 5 is not degradable—the hydrolysis of a single esterbond in the side chain reduces the net charge of a polymer chain by two,but the polymer chain itself is not cleaved. As such, films fabricatedfrom polymer 5 would likely remain stable in physiological media longerthan films fabricated from degradable polyamines, and erode or releaseDNA only after a threshold number of side chains esters are cleaved.This view is supported by the observation of lag phases in the releaseand erosion profiles shown in FIGS. 9A and 9B.

Additionally, a consideration important with respect to the applicationof these materials to promote localized or surface-mediated celltransfection is the structural and functional integrity of the plasmidDNA that is released. Cell transfection experiments using samples of DNAcollected at various times during the erosion of a polymer 5/DNA filmand a commercially available cationic lipid transfection agent.Fluorescence micrographs of COS-7 cells were obtained 48 h aftertreatment with samples of DNA collected over three periods ranging from˜16 to 27 days, 48 to 59 days, or 70 to 80 days. These micrographsdemonstrated that the DNA released over these extended time periodsremained capable of mediating high levels of expression of EGFP inmammalian cells. The structural integrity of the DNA released over thesetime periods was also examined using agarose gel electrophoresis. Theseexperiments demonstrated that a significant fraction of DNA was releasedas supercoiled DNA (e.g., from 30% to 50%), with the remainder beingreleased in an open circular topology. These results contrastsignificantly with those of past studies of the release of DNA frommultilayered assemblies fabricated from degradable poly(β-amino ester)s,for which DNA is released almost entirely in an open circular form. [J.Zhang, L. S. Chua, D. M. Lynn, Langmuir 2004, 20, 8015.]

Characterization of solutions of released DNA by dynamic lightscattering demonstrated the presence of aggregates ranging in size from˜100 to 600 nm. The zeta potentials of these aggregates were measured tobe negative (−11.3 mV). However, these values were less negative thanzeta potentials measured for solutions of naked plasmid DNA (−29.2 mV).These results indicated that the DNA released from polymer 5/DNA filmsmay be released in a form that is at least partially associated withpolymer 5.

Materials. Poly(2-vinyl-4,4-dimethylazlactone) (polymer 4, Mn=49,800,PDI=4.3) is prepared by art known methods. 3-Dimethylamino-1-propanol,3-dimethylamino-1-propylamine, and 1,8 diazabicyclo[5.4.0]undec-7-ene(DBU) were obtained from Acros Organics. Sodium acetate buffer waspurchased from Aldrich Chemical Company (Milwaukee, Wis.). Test graden-type silicon wafers were purchased from Si-Tech, Inc. (Topsfield,Mass.). Phosphate-buffered saline (PBS) was prepared by dilution ofcommercially available concentrate (EM science, Gibbstown, N.J.).Plasmid DNA [pEGFP-N1 (4.7 kb), >95% supercoiled] was obtained from ElimBiopharmaceuticals, Inc. (San Francisco, Calif.). All materials wereused as received without further purification unless noted otherwise.Deionized water (18 MΩ) was used for washing steps and to prepare allbuffer and polymer solutions. Compressed air used to dry films andcoated substrates was filtered through a 0.4 μm membrane syringe filter.General Considerations. ¹H NMR spectra were recorded on a Bruker AC+ 300spectrometer. Chemical shift values are reported in ppm and arereferenced to residual protons from solvent. Silicon substrates (e.g.,0.5×2.0 cm) used for the fabrication of multilayered films were cleanedwith acetone, ethanol, methanol, and deionized water, and dried under astream of filtered compressed air. Surfaces were then activated byetching with an oxygen plasma for 5 minutes (Plasma Etch, Carson City,Nev.) prior to film deposition. The optical thicknesses of filmsdeposited on silicon substrates were determined using air-dried filmsand a Gaertner LSE ellipsometer (632.8 nm, incident angle=70°). Datawere processed using the Gaertne Ellipsometer Measurement Program.Relative thicknesses were calculated assuming an average refractiveindex of 1.58 for the multilayered films. Thicknesses were determined inat least four different standardized locations on each substrate and arepresented as an average (with standard deviation) of independentmeasurements made on three separate films. UV-visible absorbance valuesfor PBS solutions used to determine film release kinetics were recordedon a Beckman Coulter DU520 UV/vis Spectrophotometer (Fullerton, Calif.).Film topography and surface roughness of air-dried films were obtainedfrom height data imaged under air in tapping mode on a NanoscopeMultimode atomic force microscope (Digital Instruments, Santa Barbara,Calif.). Silicon cantilevers with a spring constant of 40 N/m and aradius of curvature of less than 10 nm were used (model NSC15/Al BS,MikroMasch USA, Inc., Portland, Oreg.). For each sample, at least twodifferent scans were obtained at randomly chosen points near the centerof the film at each time point. Height data were flattened using a2nd-order fit. Root-mean squared surface roughness (Rrms) was calculatedover the scan area using the Nanoscope® IIIa software package (DigitalInstruments, Santa Barbara, Calif.).

Synthesis of Polymer 5 and 6. Polymers 5 and 6 were prepared by reactingpoly(2-vinyl-4,4′-dimethylazlactone) (4) with hydroxyl or primary aminefunctionalized nucleophiles. [23 Jewell and Lynn 2008] For the synthesisof polymer 5: polymer 4 (2 mmol), 3-dimethylamino-1-propanol (3 mmol),and DBU (0.2 mmol) were weighed into a vial and dissolved in THF (2.0mL). The reaction mixture was sealed, heated to 60° C., and stirred for24 hrs. The resulting reaction products were concentrated in vacuo andprecipitated into a hexane and acetone mixture (1:1, v/v) containing 2mmol of HCl. The precipitate was then dissolved in methanol andreprecipitated twice more. The final product was dried under vacuum toyield off-white flakes. ¹H NMR data for polymer 5: (D₂O, 300.135 MHz) δ(ppm)=4.23 (br t, 2H); 3.15 (br, t, 2H); 2.80 (br m, 7H); 2.10 (br m,2H); 1.50 (br m, 8H).

For the synthesis of polymer 6: polymer 4 (0.7 mmol) and3-dimethylamino-1-propylamine (1.1 mmol) were weighed into a vial anddissolved in THF (2.0 mL) and the mixture was subsequently heated to 50°C. in an oil bath. After 7 hours, the resulting reaction products wereconcentrated in vacuo, dissolved in methanol, and precipitated into ahexane and acetone mixture (1:1, v/v) containing 1.5 mmol of HCl. Theprecipitate was isolated by centrifugation, dissolved in methanol, andreprecipitated twice more. The final product was dried under vacuum toyield light yellow flakes. ¹H NMR data for polymer 6: (D₂O, 300.135 MHz)δ (ppm)=3.20 (br m, 5H); 2.89 (s, 6H); 1.95 (br m, 4H); 1.52 (br m, 6H).

Characterization of Kinetics of Ester Hydrolysis. ¹H NMR experimentsdesigned to characterize the loss of ester functionality in polymer 5 inaqueous solution were performed in the following manner. Polymer 2 (10mg) was dissolved in deuterated phosphate buffer (1.0 mL, 0.5 M,pH=7.2). 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (2 mg)was added as an internal standard, and this solution was transferred toa glass NMR tube. The NMR tube was incubated at 37° C. and removedperiodically for analysis by ¹H NMR spectroscopy. The change of theresonance corresponding to the methylene protons adjacent to the esterfunctionality (at 4.2 ppm) to a resonance at 3.7 ppm after hydrolysiswas monitored, and the extent of hydrolysis was determined byintegrating these signals versus the trimethylsilyl protons of theinternal standard.

Fabrication of Multilayered Films. Solutions of polymers 5 and 6 (5 mMwith respect to the molecular weight of polymer repeat units) and DNA (1mg/ml) used for dipping were prepared in sodium acetate buffer (100 mM,pH=5.1). Multilayered films were fabricated on planar silicon substratesmanually using an alternating dipping procedure according to thefollowing general protocol: 1) Substrates were submerged in a solutionof polyamine for 5 minutes, 2) substrates were removed and immersed inan initial water bath for 1 minute followed by a second water bath for 1minute, 3) substrates were submerged in a solution of DNA for 5 minutes,and 4) substrates were rinsed in the manner described above. This cyclewas repeated until the desired number of polyamine/DNA bilayers wasreached. Films were either used immediately or dried under a stream offiltered, compressed air and stored in a vacuum dessicator until use.All films were fabricated at ambient room temperature.

Characterization of Film Erosion and Release Kinetics. Experimentsdesigned to investigate film erosion and DNA release kinetics wereperformed in the following general manner: Film-coated substrates wereplaced in a plastic UV-transparent cuvette and 1.0 mL of phosphatebuffered saline (PBS, pH=7.4, 137 mM NaCl) was added to cover thefilm-coated portion of the substrate. The samples were incubated at 37°C. and removed at predetermined intervals to be characterized byellipsometry or atomic force microscopy (AFM). Films were rinsed underdeionized water and dried under a stream of filtered compressed airprior to measurement. Values were determined in at least four differentpredetermined locations on the substrate by ellipsometry and the samplewas returned immediately to the buffer solution. For experimentsdesigned to monitor the concentration of DNA in the solution, a UVabsorbance reading at 260 nm was made on the solution used to incubatethe sample.

For plasmid release experiments designed to produce samples for celltransfection experiments, erosion experiments were conducted as abovewith the following exceptions: at each predetermined time intervalsubstrates were removed from the incubation buffer, placed into a newcuvette containing fresh PBS, and the original plasmid-containingsolution was stored for analysis.

Agarose Gel Electrophoresis Assays. Samples of plasmid DNA collectedfrom film erosion experiments were evaluated by loading 30 μL of plasmidsolution into 1% agarose gels (HEPES, 20 mM, pH=7.2, 108V, 45 min).Samples were loaded on the gel with 2 μL of a loading buffer consistingof 50/50 glycerol water (v/v). DNA bands were visualized by ethidiumbromide staining, and relative intensities of bands corresponding tosupercoiled and open circular DNA were determined using Image J.Assignment of bands was aided by restriction enzyme digestion ofrecovered DNA samples by digestion by Not I and by digestion by NotI andBamHI.

Cell Transfection Assays. COS-7 cells were grown in 96-well plates at aninitial seeding density of 15,000 cells/well in 200 μL of growth medium(90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum,penicillin 100 units/mL, streptomycin 100 μg/mL). Cells were grown for24 hours, at which time the 50 μl of a Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.) and plasmid mixture was added directly to the cellsaccording to the general protocol provided by the manufacturer. TheLipofectamine 2000/plasmid transfection milieu was prepared by mixing 25μl of the plasmid solution collected at each time point during releaseexperiments (arbitrary concentrations but constant volumes) with 25 μlof diluted Lipofectamine 2000 reagent (25 μL stock diluted into 975 μLof water). Fluorescence microscopy images were acquired after 48 hoursusing an Olympus IX70 microscope and analyzed using the Metavue version4.6 software package (Universal Imaging Corporation).

While certain specific embodiments have been illustrated and described,it should be understood that changes and modifications can be madetherein in accordance with ordinary skill in the art without departingfrom the invention in its broader aspects as defined in the followingclaims.

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1. A polyelectrolyte assembly comprising at least one anion/cationicpolymer bilayer where the cationic polymer is a cationic polymerselected from: (a) a polymer of formula I:

where: n, m, l, k and i are zero or integers where n+m+l+k+i=N, thetotal number of repeat units in the polymer; A₁₋₃, B₁₋₃, C₁₋₃, D₁₋₃ andE₁₋₃ are linkers which may be the same or each may be different; Z₁-Z₅are most generally covalent bonds which may or may not be degradablebonds; R₁₋₄ and R₁₁₋₁₉, independently, can be hydrogen, or alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups,all of which are optionally substituted, with the exception that R₁₋₄are not esters; and R₅-R₁₀ are linkers or covalent bonds which may bethe same or each may be different; or (b) a polymer of formula II:

where n+m=N is the number of repeating units in the polymer; each y,independently, is 1, 2 or 3; each x, independently, is an integerranging from 1-10; each R¹, each R², each R³ and each R⁴, independently,is selected from the group consisting of hydrogen, alkyl groups, alkenylgroups, alkynyl groups, carbocyclic groups, heterocyclic groups, arylgroups, heteroaryl groups, ether groups, all of which may be substitutedor unsubstituted.
 2. The polyelectrolyte assembly of claim 1 wherein thepolymer of formula I has the formula:

where each r is an integer ranging from 1-10.
 3. The polyelectrolyteassembly of claim 2 comprising one or more different anion/cationicpolymer bilayers where at least a first bilayer is formed from acationic polymer of formula I and at least a second bilayer is formedfrom a different cationic polymer of formula I.
 4. The polyelectrolyteassembly of claim 3 wherein the cationic polymers of formula I differfrom each other in the value of (m+

)/N.
 5. The polyelectrolyte assembly of claim 4 wherein one of thecationic polymers of formula I has a value of (m+

)/N of 0.25 or less and the other cationic polymer of formula I has avalue of (m+

)/N of 0.5 or more.
 7. The polyelectrolyte assembly of claim 4 whereinone of the cationic polymers of formula I has a value of (m+

)/N of 0.25 or less and the other cationic polymer of formula I has avalue of (m+

)/N of 0.75 or more.
 8. The polyelectrolyte assembly of claim 4 whereinone of the cationic polymers of formula I has a value of (m+

)/N of 0.25 or less and the other cationic polymer of formula I has avalue of (m+

)/N of 1.0.
 9. The polyelectrolyte assembly of any of claim 2, whereinin the cationic polymers of formula I r is an integer from 1 to 3,inclusive, and each R₃ is a hydrogen or a C1-C3 alkyl group, and eachR₁₁, R₁₂ and R₁₃ is a (C1-C6) alkyl group.
 10. The polyelectrolyteassembly of claim 1, further comprising one or more additionalanion/cationic polymer bilayers wherein the one or more additionalbilayers are formed by one or more cationic polymers of formula Iwherein each cationic polymer of formula I in the assembly differs fromeach other cationic polymer of formula I in the assembly in the value of(m+l)/N.
 11. The polyelectrolyte assembly of claim 1 which comprises atleast one cationic polymer of formula I wherein the value of (m+

)/N is 0.25 or more.
 12. The polyelectrolyte assembly of claim 1 whichcomprises at least one cationic polymer of formula I wherein the valueof (m+

)/N is 0.25 or less.
 13. The polyelectrolyte assembly of claim 1 whichcomprises at least one cationic polymer of formula I wherein the valueof (m+

)/N is 0.25 or less and at least one cationic polymer of formula Iwherein the value of (m+

)/N is 0.50 or more.
 14. The polyelectrolyte assembly of claim 1, whichcomprises only cationic polymer of formula I.
 15. The polyelectrolyteassembly of claim 1 which comprises only cationic polymers of formulaII.
 16. The polyelectrolyte assembly of claim 15 comprising two or moredifferent anion/cationic polymer bilayers where at least a first bilayeris formed from a cationic polymer of formula II and at least a secondbilayer is formed from a different cationic polymer of formula II. 17.The polyelectrolyte assembly of claim 16 wherein the cationic polymersof formula II differ from each other in the value of (n)/(n+m).
 18. Thepolyelectrolyte assembly of claim 17 wherein at least one of thecationic polymers of formula II has a value of (n)/(n+m) of 0.5 or more.19. The polyelectrolyte assembly of claim 18 wherein at least one of thecationic polymers of formula II has a value of (n)/(n+m) of 0.5 or less.20. The polyelectrolyte assembly of claim 15, wherein in the cationicpolymers of formula II, all R² and R⁴ are hydrogens, all R¹ are C1-C3alkyl groups and each R³ is independently hydrogens or C1-C3 alkylgroups.
 21. The polyelectrolyte assembly of claim 1 comprising two ormore different anion/cationic polymer bilayers where at least a firstbilayer is formed from a cationic polymer of formula I and at least asecond bilayer is formed from a different cationic polymer of formulaII.
 22. The polyelectrolyte assembly of claim 21 wherein the at leastone cationic polymer of formula I has a value of (m+

)/N is 0.50 or more.
 23. The polyelectrolyte assembly of claim 21wherein the at least one cationic polymer of formula II has a value ofn/n+m of 0.5 or more.
 24. The polyelectrolyte assembly of claim 23wherein the at least one cationic polymer of formula I has a value of(m+

)/N is 0.50 or more.
 25. The polyelectrolyte assembly of claim 21,wherein in the cationic polymers of formula I, r is an integer from 1 to3, inclusive, and each R₃ is a hydrogen or a C1-C3 alkyl group, and eachR₁₁, R₁₂ and R₁₃ is a (C1-C6) alkyl group.
 26. The polyelectrolyteassembly of claim 21, wherein in the cationic polymers of formula II,all R² and R⁴ are hydrogens, all R¹ are C1-C3 alkyl groups and each R³is independently hydrogens or C1-C3 alkyl groups.
 27. Thepolyelectrolyte assembly of claim 1, wherein the anions of the bilayersare nucleic acids.
 28. The polyelectrolyte assembly of claim 1, whereinthe anions of the bilayers are nucleic acids which encode polypeptides.29. The polyelectrolyte assembly of claim 1, wherein the polyelectrolyteassembly is formed on a substrate.
 30. The polyelectrolyte assembly ofclaim 1, wherein the polyelectrolyte assembly is formed on one or mroesurfaces of an implantable medical device.
 31. A method for controllingthe release of two or more anions from a film which comprises the stepof forming a polyelectrolyte assembly of claim 1, wherein thepolyelectrolyte assembly comprises anion/cationic polymer bilayershaving two or more different anions and subjecting the polyelectrolyteassembly formed to conditions in which ester functions in the polymer offormula I and in the polymer of formula II are hydrolyzed to release thetwo or more anions.
 32. The method of claim 31 wherein the two or moreanions are released from the polyelectrolyte assembly with separate,distinct or separate and distinct release profiles.
 33. The method ofclaim 31 wherein the two or more anions are released from thepolyelectrolyte assembly such that at least one anion is releasedshort-term and at least one anion is released long-term.